Alpha 2-macroglobulin and tissue inhibitor of metalloproteinases: collagenase inhibitors in human preovulatory ovaries.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular development.

Rats were pretreated with oestradiol-17 beta or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h. Similarly, PMSG-cells secreted 16-fold more 20 alpha-dihydroprogesterone than did E2-cells, in the absence of FSH, and 6- to 10-fold more of the 20 alpha-reduced metabolite in the presence of FSH. In contrast, fibronectin secretion by PMSG-cells was only about 60% and 30% that by E2-cells at 24 and 48 h, respectively. Fibronectin secretion by E2- and PMSG-cells was reduced in the presence of FSH at 24 and at 48 h of culture. While cells in all treatment groups underwent spreading during culture to become elongated and irregular in outline, elevated fibronectin secretion in vitro was accompanied by enhanced cellular spreading. At 24 and 48 h of culture respectively, the mean area occupied by E2-cells on the culture surface was x 2 and x 1.6 that by PMSG-cells. Coincident with its inhibitory effect on fibronectin secretion, FSH reduced the mean area occupied by E2- and PMSG-cells on the culture surface at both time intervals. These findings suggest that granulosa cell fibronectin secretion is a feature of early follicular development. It may be that the secretion of this adhesive glycoprotein by granulosa cells provides a pool of fibronectin which is used for basement membrane deposition during follicular growth.

Isolation and characterization of rabbit ovarian surface epithelium, granulosa cells, and peritoneal mesothelium in primary culture.

Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplemented D-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17 beta-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3 beta hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro.

Use of molecular probes to study regulation of aromatase cytochrome P-450.

Aromatase, an enzyme complex localized in the endoplasmic reticulum of estrogen-producing cells, is composed of NADPH-cytochrome P-450 reductase, and aromatase cytochrome P-450 (cytochrome P-450AROM). To define the molecular mechanisms involved in the multifactorial regulation of cytochrome P-450AROM in estrogen-producing cells, we have isolated a cDNA specific for human cytochrome P-450AROM and have used this cDNA to isolate the human cytochrome P-450AROM gene. The cDNA sequence encodes a polypeptide of 503 amino acids and contains--near the carboxy-terminus, a region of high homology with the putative heme-binding regions of other P-450 cytochromes. COS1 cells transfected with an expression plasmid containing the cytochrome P-450AROM cDNA had the capacity to aromatize testosterone, androstenedione and 16 alpha-hydroxyandrostenedione, suggesting that a single polypeptide catalyzes all steps of the aromatization reaction using either of the three major C19-substrates. The human cytochrome P-450AROM gene is greater than 52 kb in size and consists of 10 exons and 9 introns. Hormonally induced changes in aromatase activity of human ovarian granulosa and adipose stromal cells are associated with comparable changes in cytochrome P-450AROM gene expression and synthesis, whereas the reductase component is only modestly affected. Studies are in progress to define the molecular mechanisms involved in the regulation of cytochrome P-450AROM gene expression in estrogen-producing cells.

Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles.

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.

Studies on follicular atresia: role of gonadotropins and gonadal steroids in regulating cathepsin-D activity of preovulatory follicles in the rat.

Preovulatory follicular atresia was studied using pregnant mare serum gonadotropin (PMSG)-primed rats (15 IU/rat) which were deprived of hormonal support either by allowing the metabolic clearance of the PMSG or by injecting a specific PMSG antiserum (PMSG a/s). Atresia was monitored by an increase in lysosomal cathepsin-D activity and a decrease in the receptor activity of the granulosa cells (GC) isolated from the preovulatory follicles. It was shown that the increase in lysosomal activity and the decrease in receptor activity seen at 96 h after PMSG (or PMSG plus PMSG a/s) could be arrested both by follicle stimulating hormone (FSH) and luteinizing hormone (LH). Injection of cyanoketone or clomiphene citrate together with FSH/LH prevented this 'rescue' suggesting a role for estrogens in the regulation of atresia. Although the administration of estradiol-17 beta (20 micrograms/rat) together with PMSG a/s could show a 'rescue effect' in terms of reduction in cathepsin-D activity the gonadotropin receptor activities of these granulosa cells were not restored. The injection of dihydrotestosterone (DHT) to 48 h PMSG-primed rats induced atresia as noted by an increase in cathepsin-D activity. However, the exogenous administration of FSH along with DHT prevented this atretic effect suggesting that DHT is not having a direct effect on atresia. Determination of androgen: estrogen content of the granulosa cells and an analysis of the individual profile of androgen and estrogen revealed that the increase in cathepsin-D activity could be correlated only with the decrease in GC estrogen content. This along with the observation that GC showed a loss of estrogen synthesis well before the increase in cathepsin-D activity strongly points out that the lack of estrogen rather than an increase in androgen is the principle factor responsible for the atresia of preovulatory follicles in the rat.

Lectin binding sites of cultured ovarian Sertoli cell tumors and follicular granulosa cells.

A panel of seven alkaline phosphatase labeled lectins was used to probe nitrocellulose electroblots of SDS-PAGE separated proteins from a primary culture of normal ovarian granulosa cells and an ENU-induced Sertoli cell tumor cell line (SCTL-I). Several additional lectin binding proteins were observed in silver stained SDS-PAGE gels as well as with lectins in SCTL-I. Succinated concanavalin A (Suc. Con A), Ricin communis agglutinin (RCA-I), Ulex europaeus agglutinin (UEA 1), Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA) and Peanut agglutinin (PNA) stained more intensely in SCTL-I than normal granulosa cells. The same lectins as above, labeled with fluorescein isothiocyanate (FITC), were used to study the distribution of specific binding sites of tissue cultured cells grown in chamber/slides. Both normal ovarian granulosa cells and SCT cells exhibited strong peninuclear cytoplasmic labeling with Con A UEA-1 and WGA exhibited predominantly a nuclear and granular cytoplasmic staining pattern. SBA and DBA exhibited a strong coarse granular cytoplasmic labeling in granulosa cells and moderate granular cytoplasmic in SCT cells. In granulosa cells, Golgi regions stained strongly with PNA but weakly in SCT cells. RCA-I staining was negative in both cultures. Labeling of tissue cultured cells with lectins provides more details than histological sections of lectins binding sites at cellular structural levels.

Changes in cytosolic free calcium ion concentrations in individual rat granulosa cells: effect of luteinizing hormone-releasing hormone.

Changes in the cytosolic free calcium ion concentrations ([Ca2+]i) induced by LHRH were studied in individual rat granulosa cells using fura-2 microspectrofluorimetry. The resting [Ca2+]i concentration was 96.7 +/- 2.9 nM (n = 115). The majority of the granulosa cells responded to LHRH at doses between 10(-7) and 10(-5) M; the latter dose was the highest concentration required to initiate alterations in [Ca2+]i in these cells. The alterations in [Ca2+]i induced by LHRH were transient and returned to resting levels within 84 +/- 3 sec (n = 64). A potent LHRH antagonist completely blocked the effect of LHRH on [Ca2+]i. Within a single granulosa cell, three consecutive injections of the same dose of LHRH, delivered 5 min apart, induced three discrete peaks of [Ca2+]i of similar amplitudes. Sustained perfusions of LHRH, however, resulted in a desensitization of the [Ca2+]i response to LHRH, but not to the calcium ionophore Br-A23187. These results, which were obtained from individual cells, provide strong support for the hypothesis that acute changes in [Ca2+]i are involved in the early cellular transduction of the LHRH signal in the ovary.

Human chorionic gonadotropin localization and morphometric characterization of human granulosa-luteal cells obtained during in vitro fertilization cycles.

The area and cytoplasmic-to-nuclear ratio (C/N) of cells aspirated from follicles with mature oocytes was determined using a computerized image analysis system. The presence of human chorionic gonadotropin (hCG) on the surface membrane and/or within the cytoplasm of each cell also was determined using a horseradish peroxidase immunocytochemical procedure. Based on morphometric characteristics, follicular cells were classified as granulosa or luteal. Granulosa cells were less than 75 micron 2 in area with a C/N of approximately 0.5. Luteal cells were classified as small (less than 75 micron 2, C/N approximately 1.5), midluteal (76 to 100 micron 2, C/N greater than 1.5) and large luteal (greater than 100 micron 2, C/N greater than 1.5). Compared with aspirates from follicles containing fertilizable oocytes, aspirates from follicles with nonfertilizable oocytes had fewer granulosa cells and more large luteal cells. HCG was localized on the membranes of granulosa and small luteal cells and within the cytoplasm of midluteal cells. Human chorionic gonadotropin was generally not observed on either the membranes or cytoplasm of luteal cells over 120 micron 2. These data support the concept that granulosa cells bind hCG to membrane receptors, internalize hCG, and begin to luteinize in response to hCG stimulation. Since the aspirates from follicles containing nonfertilizable oocytes possessed a higher percentage of large luteal cells, it is postulated that the cells from these aspirates began the luteinization process earlier than those from follicles containing fertilizable oocytes.

Physiologic characterization of transformed and cloned rat granulosa cells.

Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions.

Oestrogen receptor mRNA and a related RNA transcript in mouse ovaries.

Oestrogen receptor mRNA expression in mouse ovaries was analysed by Northern blotting of total RNA using 32P-labelled RNA probes complementary to different functional domains of the oestrogen receptor. The approximately 6.5 kb mouse oestrogen receptor mRNA transcript was present in immature and adult ovaries at extremely low abundance compared with uterus and oviduct. Using a probe complementary to the steroid-binding domain of the oestrogen receptor (probe EF), a novel RNA transcript of approximately 1.5 kb was also found in the ovaries but was absent from uterus and oviduct. The melting temperature of the hybrid produced by the approximately 1.5 kb transcript with probe EF was approximately 10 degrees C lower than that produced by authentic oestrogen receptor mRNA, which demonstrates incomplete sequence homology between the two transcripts and indicates that the approximately 1.5 kb RNA is not a truncated form of oestrogen receptor mRNA. Furthermore, the approximately 1.5 kb RNA lacks the DNA-binding domain found in the oestrogen receptor. The approximately 1.5 kb RNA, but not oestrogen receptor mRNA, was enriched in total RNA from isolated granulosa cells compared with residual ovarian tissue. The encoded product of this novel oestrogen receptor-related RNA could be a steroid-binding protein involved in oestrogen action in the ovaries.

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development.

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.

GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells.

Gonadotropin-releasing hormone (GnRH)-like proteins with anti-gonadotropic properties were recently discovered in the ovaries of several species, including humans. Since neither GnRH receptors nor GnRH are in bovine ovarian tissue, we examined, in the present studies, whether concentrations of GnRH-like proteins varied during development of the corpus luteum (CL) and whether GnRH-like proteins were selectively localized in ovarian cells of cows. For these studies, GnRH-like proteins were extracted from various ovarian and nonovarian tissues and fluids and fractionated for hydrophobic interaction chromatography. A highly specific and sensitive radioreceptor assay (RRA) was used to quantify concentrations of GnRH-like proteins. The major findings of these studies demonstrated that 1) the amount of GnRH-like proteins in the corpus luteum (CL) was proportional to the weight of the CL; 2) the concentration of GnRH-like proteins in luteal tissue decreased during development of the CL; 3) GnRH-like proteins were in ovarian and numerous nonovarian tissues, but were not in the heart, plasma, or follicular fluid; 4) the retention time for GnRH-like proteins following high-pressure liquid chromatography (HPLC) varied with the tissue source; and 5) compared with all other tissues, the greatest concentration of GnRH-like proteins was in granulosal cells. We concluded that the concentration of GnRH-like proteins in luteal cells decreased during development of the CL, and that a specific GnRH-like protein was selectively localized in bovine granulosal cells.

Immunocytochemistry of ion transport mediators in the genital tract of female rodents.

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.

Inhibin: studies of stored and secreted forms by biosynthetic labeling and immunodetection in cultured rat granulosa cells.

The biosynthesis of inhibin in rat granulosa cells was studied by biosynthetic labeling, immunoblotting, and immunocytochemical techniques. Granulosa cells from immature hypophysectomized estrogen-treated rats were cultured in the presence of [35S]cysteine. Both conditioned media and cell extracts were subjected to immunoprecipitation with an antibody directed against the N-terminal 26 amino acids of the alpha-chain of porcine inhibin (pI alpha 1-26), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Treatment with FSH (100 ng/ml) and delta 4-androstenedione (10(-7) M) increased the secretion of 35S-labeled inhibin immunoreactivity by 2.6-fold over that in control cultures treated with androstenedione alone. The radiolabeled inhibin had mol wt (Mr) values of 45,000 and 30,000. Upon reduction, the 45,000 Mr polypeptide remained (with increased apparent Mr of 49,000), but the 30,000 Mr species disappeared with the concomitant appearance of two bands with 18,000 and 11,000 Mr. Competition studies with pI alpha 1-26 confirmed that these polypeptides were all related to inhibin. Furthermore, immunoblotting with an antibody directed against the porcine inhibin beta-A chain (pI beta A81-113) indicated that the 11,000 Mr peptide was the inhibin beta-A chain. Extracts of cells treated with FSH contained only a high Mr alpha-related species (Mr, 41,000 nonreduced; 49,200 reduced). The inhibin alpha antibody was also used to immunocytochemically stain cultured granulosa cells. Cells that had been treated with FSH or the adenyl cyclase activator forskolin (3 x 10(-5) M), but not untreated cells, exhibited positive staining. These results indicate that granulosa cells synthesize and store inhibin alpha-chain precursor with 49,000 Mr. Although some of the high Mr alpha-form was secreted, the majority of the alpha-subunit was processed to the 18,000 Mr form and dimerized with the 11,000 Mr beta-chain to form the mature inhibin dimer immediately before secretion. The cultured granulosa cells may provide a model for future studies on the hormonal regulation of inhibin alpha- and beta-gene expression as well as subunit dimerization and secretion.

Flow cytometric analysis of deoxyribonucleic acid in human granulosa cells from in vitro fertilization cycles: relationships to oocyte maturity and fertilizability and to follicular fluid steroids.

We measured the mitotic activity of granulosa cells, sex steroid concentrations in follicular fluids, and the maturity and fertilizability of oocytes from 49 follicles. Flow cytometric measurements of DNA were used to determine the percentage of cells in G0/G1, S, and G2/M phases of the cell cycle. Mitotic index was designated as the percentage of granulosa cells in S + G2/M. The progesterone concentration and the progesterone to estradiol ratio in follicular fluids were inversely correlated to mitotic index (r = -0.506; P less than 0.001, and r = -0.320; P less than 0.02, respectively). Estradiol and androstenedione levels did not correlate with the mitotic index. The mitotic index was higher in follicles with immature oocytes [25.6 +/- 2.0% (+/- SE); n = 7] than in follicles with mature oocytes (15.6 +/- 1.2%; n = 41; P less than 0.001). The mitotic index of granulosa cells was lowest in follicles with oocytes that fertilized (15.5 +/- 1.8%), higher in follicles with oocytes that remained unfertilized (18.5 +/- 1.3%; P less than 0.03), and highest in follicles with oocytes that fertilized abnormally (24.0 +/- 2.1%; P less than 0.02). Differences in maturity or fertilizability of oocytes were not associated with variations in follicular fluid progesterone concentrations. The study supports the concept that mitotic activity is decreased when granulosa cells become luteinized. During early follicular growth it is assumed that estradiol and perhaps androstenedione may be important regulators of cell division. Our findings suggest that progesterone, perhaps acting as an antiestradiol, is more important in controlling granulosa cell division of preovulatory follicles during the late follicular phase.

Immunocytochemical localization of progesterone receptor in the chick ovary.

The distribution of progesterone receptor (PR) in the chick ovary was studied using light- and electron-microscopic immunocytochemical methods. With the light microscopic technique, PR was observed in the germinal epithelium cells of estrogen-treated and estrogen-untreated immature chicks. With the preembedding immunocytochemical technique, which proved to be more sensitive than immunohistochemistry using paraffin sections, the germinal epithelium and also part of the thecal and stromal cells were stained when immature chicks were not treated with estrogen. After estrogen treatment, the number of PR-positive stromal and thecal cells increased, as did the immunostaining intensity in their nuclei. The granulosa cells were PR-positive only after estrogen treatment. In the ovary of laying hens, the most intense staining for PR was localized in the germinal epithelium. PR was also present in the thecal, stromal, and granulosa cells. At the subcellular level, PR was detected only in the cell nuclei, indicating that ovarian PR is intranuclear independent of the presence of progesterone. In conclusion, immunocytochemical methods proved to be suitable for studying steroid hormone receptors in steroid-producing tissues (e.g. the ovary), because excess endogenous hormones do not affect detection of the receptor with the antibody as they do detection with labeled ligands. Immunocytochemically, the germinal epithelium, stromal, thecal, and granulosa cells of the laying hen ovary were shown to be target cells for progesterone. The inducibility of PR by estrogen in the thecal, stromal, and granulosa cells suggests that these cell types are also sensitive to estrogen.

Changes in catecholamine contents in ovarian follicular theca, progesterone contents in granulosa, and LH concentrations in peripheral plasma during the ovulatory cycle in the hen.

By the use of high-performance liquid chromatography, the content of norepinephrine (NE) and epinephrine (E) in the theca of the largest and the second largest preovulatory follicle of the ovary of the hen was found to increase from 15 h (NE) or 18 h (E) to reach a peak 9 h (NE) or 9-6 h (E) before ovulation of the largest follicle. The content of progesterone in the granulosa of these follicles and the concentration of LH in the peripheral plasma of the same hen, both of which were measured by radioimmunoassays, showed a peak 6 h before ovulation. The results suggest that the catecholamines are involved in some events occurring in both follicles some several hours before ovulation of the largest follicle during the ovulatory cycle.

Presence of gonadotropin-releasing hormone-like proteins in bovine and ovine ovaries.

Recently, the rat ovary was shown to contain significant levels of a GnRH-like protein, but no detectable GnRH. In the present studies, extracts of bovine ovaries and ovine corpora lutea were examined for their content of both GnRH-like protein and GnRH. The GnRH-like proteins were detected with a rat ovarian membrane radioreceptor assay, and GnRH was detected with a specific GnRH RIA. The biological activity of the GnRH-like protein was evaluated with a rat luteal cell assay. GnRH-like activity, but not GnRH, was clearly present in extracts of the entire bovine ovary, bovine corpus luteum, bovine granulosa cells, and ovine corpus luteum. The highest levels of GnRH-like activity were present in granulosa cells. Neither GnRH-like activity nor GnRH was detected in extracts of bovine follicular fluid or bovine jugular plasma. Fractionation of the bovine and ovine GnRH-like proteins by reverse phase HPLC resulted in retention times similar to that of the rat ovarian GnRH-like protein, but distinctly different from that of authentic GnRH. The bovine and ovine GnRH-like fractions, like those in the rat, were sensitive to protease and heat. The bovine GnRH-like protein, obtained by preparative reverse phase HPLC, evoked a dose-dependent inhibition of LH-stimulated cAMP accumulation in rat luteal cells similar to that caused by GnRH. Based on these results, we suggest that the bovine ovary, granulosa cells, and corpus luteum and the ovine corpus luteum contain an antigonadotropic GnRH-like protein similar to the GnRH-like protein of the rat ovary. The presence of a similar GnRH-like protein (but the absence of GnRH) in ovaries of domestic species and the rat raises the possibility that this substance may play a paracrine antigonadotropic role in the ovary of diverse species.