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Biology of Reproduction 59, 836-842 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Developmental Changes in the Expression of the Growth Hormone Receptor Messenger Ribonucleic Acid and Protein in the Bovine Ovary1

Sabine Kölle2,a, Fred Sinowatza, Gudrun Boiea, , and David Lincolnb

a Department of Veterinary Anatomy, University of Munich, 80539 Munich, Germany b Faculty of Allied Health Sciences, University of Kuwait, 90805 Sulaibikhat, Kuwait


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By reverse transcription-polymerase chain reaction, the transcript of the growth hormone receptor (GHR) was demonstrated in oocytes, follicular cells, and corpus luteum of the bovine ovary. Immunoblotting using the monoclonal antibody mAb 263 resulted in a distinct protein band at 120 kDa, confirming that translation of the mRNA takes place in the same cells. Nonradioactive in situ hybridization revealed that distribution of the mRNA encoding GHR was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the oocyte showed distinct amounts of the transcript encoding GHR, in tertiary follicles the mRNA was predominantly localized in the cells of the cumulus oophorus. GHR mRNA was also expressed in the large granulosa lutein cells, in the germinal epithelium, and in the endothelial cells of ovarian vessels. Colocalization of the GHR protein showed a distribution pattern identical to that of the mRNA. In calves, oocyte and follicle cells changed GHR expression in the same way as in the adult ovary. During embryonic development of the ovary, distinct amounts of the mRNA encoding GHR were found in primordial follicles shortly before birth. Our results imply that the GHR is involved in ovarian ontogenesis, especially in early folliculogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. GH-deficient animals and humans reveal a delay in the onset of puberty, which can be induced by GH treatment [1, 2]. Additionally, GH increases the sensitivity of the human ovary to stimulation by gonadotropins. Thus, in some patients with previously poor response to gonadotropins during in vitro fertilization and embryo transfer, the dose and duration of gonadotropin application for inducing ovulation can be reduced by treatment with GH [3, 4]. In hypophysectomized ewes, growth and ovulation of follicles are not induced by exogenous gonadotropins unless GH is additionally given [5]. In vitro studies have shown that GH enhances FSH-induced differentiation of rat granulosa cells [6] and stimulates human and bovine ovarian steroidogenesis [7, 8]. Up to now, it is not known whether these effects are mediated by direct action of GH on the ovary or by changing concentrations of insulin-like growth factor-I [9].

Although GH has been shown to exert direct effects on granulosa cells [6, 10] and ovarian follicles [11, 12], there have been difficulties in localizing specific receptors for GH in follicles of domestic livestock species. Using immunohistochemistry, Lucy et al. [13] demonstrated GH receptor (GHR) in bovine large luteal cells, but no immunoreactivity was found in follicles of any size. Experiments to characterize GH binding to bovine follicular tissues by radioreceptor assay failed [14]. In 1997, Eckery et al. [5] also reported that attempts to localize receptors for GH in sheep ovarian follicles by topical autoradiography were unsuccessful. Binding sites for GH in the ovine ovary could be demonstrated only by in situ hybridization detecting the mRNA encoding GHR [5].

Up to now, human [15], rabbit [15], mouse [16], rat [17], pig [18], sheep [19], and cow [20] GH receptors have been cloned. As shown by SDS-PAGE, the bovine GHR gene encodes a protein with a molecular mass of 120 kDa [21].

The purpose of our study was to localize GHR and its transcript in the bovine ovary during embryonic development and folliculogenesis. Using reverse transcription-polymerase chain reaction (RT-PCR), nonradioactive in situ hybridization, Western blotting, and immunohistochemistry, we examined ovaries of fetuses, calves, and cows as well as cumulus-oocyte complexes (COCs), isolated oocytes, follicular cells, and follicular fluid.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Ovaries

Ovaries of 28 cows, 14 calves (mean age: 6 mo), and 16 fetuses aged from 8 wk to 9 mo (i.e., fetuses of crown-rump lengths of 5, 10, 15, 18, 21, 28, 38, 48, 51, 56, 61, 66, 70, 75, 80, 96 cm) were obtained from the local slaughterhouse. Twelve corpora lutea from nonpregnant cows (corpora lutea cyclica) and 11 corpora lutea from cows of the second until ninth month of pregnancy (corpora lutea graviditatis) were collected separately. For RT-PCR, the tissue was snap-frozen in liquid nitrogen and stored at -70°C until use. For in situ hybridization, ovaries were cut into pieces of 1 x 1 cm and fixed in 4% paraformaldehyde (PFA) in PBS for 18 h. After washing steps in 0.5 M sucrose and in PBS, specimens were snap-frozen in liquid nitrogen and embedded in tissue-freezing medium (Shandon Southern Products Ltd., Cheshire, England). Sections (8 µm) were mounted on 3-aminopropylene-ethoxysilane-coated slides, dried at 40°C for 15 min, and stored at -70°C until use.

For Western blotting, 0.2 g of ovarian cortex and corpus luteum was snap-frozen in liquid nitrogen and homogenized in 350 µl 0.005 M Tris-HCl, pH 7.2. After centrifugation at 22 000 x g, the supernatant was frozen at -20°C. For immunohistochemistry, pieces of ovarian tissue were fixed in Bouin's fluid overnight, dehydrated in a graded series of ethanol, and embedded in paraffin. Sections (5 µm) were cut on a Leitz (Leitz Wetzlar GBH, Wetzlar, Germany) microtome.

Preparation of COCs, Follicular Fluid, Follicular Cells, and Oocytes

COCs were obtained by aspiration of 2- to 8-mm follicles of fresh ovaries using a 20-gauge needle and a 2-ml syringe. The COCs were collected with a transfer pipette under microscopic control and washed in PBS several times. They were dried on coated glass slides at 50°C for 15 min, fixed in 4% PFA for 5 min, dehydrated in a graded (30–100%) series of ethanol, and frozen at -70°C until use. A portion of the COCs were fixed in Bouin's fluid, dehydrated, and embedded in paraffin. For isolation of oocytes and follicular cells, the COCs were collected in PBS. Oocytes and follicular cells were mechanically separated by vortexing for 90 sec. The oocytes were collected, washed in PBS, and transferred to Tripure Reagent (Sigma, Deisenhofen, Germany). The remaining follicular cells were homogenized in 0.005 M Tris, pH 7.2, and centrifuged. The supernatant was frozen at -20°C. The remaining fluid obtained by follicular aspiration was centrifuged at 300 x g. The supernatant containing follicular fluid without cells was frozen at -20°C.

RT-PCR

Total cellular RNA was isolated using Tripure Reagent (Sigma) according to the manufacturer's instructions. The RT reaction mix (total volume: 20 µl) included 5 µg RNA, single-strength Expand reverse transcriptase buffer (50 mM Tris-HCl, 40 mM MgCl2, 0.5% Tween 20 [v:v], pH 8.3), 10 mM dithiothreitol, 1.6 µg oligo(dT), 0.75 mM dNTP, 3 mM MgCl2, 30 units RNase inhibitor, and 50 units Expand reverse transcriptase. The PCR mix (total volume of 50 µl) contained 5 µl RT reaction, single-strength reaction buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl2, pH 8.3), 0.4 mM dNTP, 4 U Taq polymerase, and 100 pmol of each primer.

The primers for the amplification of bovine GHR cDNA were used as described by Scott et al. [22]. The forward GHR primer was 5'-ACC CAG TGG AAA ATG GAC CCT T-3'; the reverse primer was 5'-CTG TCT GTG TCT GAC CCT TCA GTC-3'.

In oocytes, the total amount of isolated RNA was diluted in 6.1 µl distilled water and 4 µl single-strength Expand reverse transcriptase buffer. Twenty microliters of the RT-reaction was used for PCR. All RT-PCR reagents were purchased from Böhringer Mannheim, Germany. The amplification conditions were 94°C denaturation for 1 min, 60°C annealing for 2 min, and 72°C extension for 3 min. A final extension at 72°C for 7 min followed after 29 cycles. The PCR product was analyzed in a 2% agarose gel and stained with ethidium bromide. The amplification product was purified and sequenced by Medigene (Martinsried, Germany).

In Situ Hybridization

Slides adjusted to room temperature were hydrated in a graded series of ethanol (100% to 50%) and washed in PBS. Ovarian tissue was permeabilized by treatment with 0.2 M HCl (10 min) and proteinase K (4 µg/ml; Sigma-Aldrich, Deisenhofen, Germany) at 37°C (20 min). After postfixation in 4% PFA in PBS, nonspecific hybridization signals were reduced by incubation in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. Between all steps, specimens were washed in PBS. All enzyme reactions and fixation steps were stopped by incubation in PBS-glycine (2 mg/L). Slides were prehybridized at 42°C for 2 h. Both the prehybridization and the hybridization were performed in humid chambers containing filter papers saturated with 4-strength sodium chloride-sodium citrate (SSC; single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) and 50% formamide. The prehybridization solution comprised 0.5 M NaCl, 12 mM Tris, pH 7.5, 1 mM EDTA, 5-strength Denhardt's solution, 0.5 mg/ml heparin, 0.5 mg/ml salmon sperm DNA, and 50% formamide. The sequence of the oligonucleotide probe was chosen according to the cDNA of bovine GHR [20]. The sequence of the biotin-labeled antisense probe was 5'-TGG TCT GTG CTC ACA TAG CC-3' (base pairs [bp] 2067–2087).

The biotinylated probes were diluted to a concentration of 20 ng/µl. Hybridization was carried out at 43°C overnight. After the slides had been washed in 0.2 SSC for 1 h, the hybridization signals were visualized using the avidin-streptavidin-biotin complex (Dako, Hamburg, Germany) according to the manufacturer's instructions. The slides were incubated in 0.05 mg/ml diaminobenzidine containing 1% H2O2 for 10 min. After washing in distilled water, the slides were dehydrated in a graded series of ethanol (50% to 100%) and xylene and mounted with Eukitt (Riedel-de-Haen, Seelze, Germany).

For controls, specificity of hybridization was monitored in control experiments carried out with the sense probe 5'-biotin-GGC TAT GTG AGC ACA GAC CA-3'. In order to check background staining, hybridizations were also performed without oligonucleotides. Bovine liver was used as control organ.

Immunoblotting

For Western blotting and immunohistochemistry, the monoclonal antibody mAb 263 (IgG K isotype) was used. It was produced by hybridoma technology from mice immunized against a human GH-affinity-purified rabbit and rat liver GH receptor as described by Barnard et al. [23]. Monoclonal antibody 263 recognizes a cross-species determinant with high affinity and does not cross-react with insulin or prolactin receptors [24]. The antibody has been validated extensively for immunohistochemical studies in rats, rabbits, and cows [21, 25, 26].

SDS-PAGE was performed according to Laemmli [27] using protein extracts of ovarian cortex, isolated follicle cells, follicular fluid, and the corpus luteum as samples. As a control, protein extracts of bovine liver were used. In liver 50 µg protein, in follicular cells and follicular fluid 70 µg protein, and in ovarian cortex and corpus luteum 100 µg protein were electrophoresed in an 8% polyacrylamide gel.

The proteins were transferred onto nitrocellulose (pore size 0.2 µm) by semi-dry blotting. Membranes were blocked in Tris-buffered saline (TBS)/double-strength Tween/2% blocking reagent (Bio-Rad, Munich, Germany) at 4°C overnight. The GHR antibody was used at a dilution of 1:500 in TBS/double-strength Tween/1% blocking reagent. The secondary antibody, a biotinylated rabbit anti-mouse IgG (Dako), was diluted to 1:500 in TBS/double-strength Tween. All washes were performed with TBS/double-strength Tween. The proteins were labeled with the avidin-streptavidin-biotin horseradish peroxidase complex (Dako; dilution 1:50 in TBS/double-strength Tween) and detected with chemiluminescence according to the manufacturer's instructions (Amersham, Braunschweig, Germany).

Immunohistochemistry

Sections were deparaffinized in xylol and rehydrated in a graded series of ethanol. Endogenous peroxidase was eliminated by incubation in 0.5% H2O2 in PBS for 30 min. Nonspecific binding was blocked with normal rabbit serum (dilution 1:20 in PBS, 1 h at room temperature). The GHR monoclonal antibody mAb 263 was used in a dilution of 1:200. The secondary antibody was biotinylated rabbit anti-mouse IgG (Dako; dilution 1:300). After incubation of the slides in avidin-streptavidin-biotin horseradish peroxidase complex (Dako) for 1 h at 20°C (dilution 1:150 in PBS-1% BSA), proteins were detected by 0.05% diaminobenzidine in PBS containing 1% H2O2.

Controls were performed by a) omission of the primary antibody, b) omission of the secondary antibody, c) replacement of the GHR antibody by preimmune mouse serum, and d) preincubation with recombinant bovine GH (Celanco Inc., Greenfield, IN) in concentrations of 1, 2, and 4 µg/100 µl PBS for 1 h at room temperature.

To verify the identity of cells in the ovarian connective tissue marked by the GHR antibody, the monoclonal mouse macrophage antibody CD68 (Dako, Glostrup, Denmark) was used. After deparaffinization, rehydration, and elimination of endogenous peroxidase, specimens were subjected to enzymatic predigestion with trypsin (0.1% trypsin/PBS supplemented with 0.1% CaCl2, 10 min, 37°C). Blocking in rabbit normal serum was followed by incubation with the primary antibody CD68 (dilution 1:100; overnight). The secondary antibody was biotinylated rabbit anti-mouse IgG (dilution 1:300, 40 min). The protein was detected using diaminobenzidine and H2O2 as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR

RT-PCR analysis of oocytes, follicular cells, and corpus luteum showed an amplified cDNA fragment of 487 bp (Fig. 1, lanes C–E). Bovine liver, which is known to be rich in GHR, also revealed a distinct band (Fig. 1, lane B). Purification and sequencing of the PCR product (Medigene) resulted in the expected cDNA fragment of bovine GHR. The negative control, in which RNA instead of DNA was used as PCR template, revealed no amplification product (Fig. 1, lane F).



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  		FIG. 1. Detection of GHR mRNA in bovine oocytes, follicular cells, and corpus luteum by RT-PCR. The amplification product has a predicted size of 487 bp. Liver known to be rich in GHR was used as control organ. Lanes A: 100-bp ladder; B: liver; C: oocytes; D: follicular cells; E: corpus luteum; F: control (RNA as template for PCR). FIG. 2. Detection of the mRNA encoding GHR by in situ hybridization during folliculogenesis of the bovine ovary. A) Primordial follicle: Distinct amounts of the GHR mRNA are found in the oocyte (x450). B) Primordial follicle: No signal is visible in the negative control using the sense oligonucleotide (x450). C) Primary follicle: The cytoplasm of the oocytes is distinctly labeled for the mRNA encoding GHR. In the late primary follicle, follicle cells also started to express the transcript (x400). D) Secondary follicle: The follicular cells, especially the cells surrounding the oocyte, are labeled for GHR (x325). E) Tertiary follicle: The transcript of GHR is mainly localized in the follicle cells of the cumulus oophorus, especially in the cells of the corona radiata (arrow) (x200). Reproduced at 74%.

In Situ Hybridization

In primordial and primary follicles, the oocytes were distinctly labeled for the mRNA encoding GHR (Fig. 2, A and C). Control sections incubated with the sense oligonucleotide regularly revealed no signal (Fig. 2B). In the late primary follicle, the follicle cells also started to express the transcript (Fig. 2C). Secondary follicles showed labeling in follicle cells, especially in the cells surrounding the oocyte (Fig. 2D). In tertiary follicles the mRNA encoding GHR was mainly localized in the cytoplasm of the follicle cells of the cumulus oophorus and the corona radiata (Fig. 2E). The oocyte merely revealed weak signals (Fig. 2E). Theca cells were regularly not labeled for GHR. Isolated COCs obtained by aspiration of tertiary follicles also showed the transcript of GHR in the cytoplasm of the surrounding follicle cells. The endothelial cells and some smooth muscle cells of ovarian vessels and the germinal epithelium displayed distinct amounts of the GHR transcript. In corpora lutea cyclica, the large granulosa lutein cells and the endothelial cells were regularly stained. During pregnancy, amount and localization of the transcript in the corpora lutea graviditatis were identical to those in corpora lutea cyclica. The expression of the transcript in corpora lutea graviditatis did not change during various stages of pregnancy. Ovaries of calves showed the same RNA distribution pattern as in the adult. Fetal ovaries did not show any labeling until the end of the third month of pregnancy. At this time endothelial cells of the ovarian vessels revealed significant amounts of the mRNA encoding GHR in their cytoplasm. In the eighth month of fetal life, oocytes of primordial and primary follicles were distinctly labeled for the mRNA encoding GHR (see Fig. 8A).

Immunoblotting

Western blotting revealed a distinct protein band in ovarian cortex (Fig. 3A, lanes A and C), corpus luteum (Fig. 3A, lane B), and isolated follicular cells (Fig. 3B, lane A). According to the molecular weight markers (Sigma-Aldrich and Bio-Rad), bovine GHR has an apparent molecular mass of 120 kDa under reducing and nonreducing conditions. In follicular fluid, normally no protein band was seen (Fig. 3B, lane B). Bovine liver used as positive control always showed a distinct protein band (Fig. 3B, lane C).



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  		FIG. 3. Immunoblot using the monoclonal antibody mAb 263 directed against GHR. A) Lane A: ovarian cortex; lane B: corpus luteum; lane C: ovarian cortex. The GHR protein band has an apparent molecular mass of 120 kDa. B) Lane A: follicular cells; lane B: follicular fluid; lane C: liver. The GHR protein synthesized in the follicular cells is not secreted into the follicular fluid. FIG. 4. Immunohistochemical localization of the GHR protein in the bovine ovary and in isolated COCs. A) Primordial follicle: The GHR was found in the cytoplasm of the oocyte (x420). B) Primary follicle: Preincubation with GH (4 µg/100 µl PBS) strongly reduced staining in the cytoplasm of the oocyte (x420). C) Late primary follicle: The oocyte is distinctly stained for GHR (x400). D) Secondary follicle: Labeling for GHR is seen in the oocyte and the granulosa cells (x325). E) Tertiary follicle (calf): The GHR protein is localized in nucleus and cytoplasm of cumulus cells. In calves the protein reveals the same localization in the ovary as in cows (x240). F) Isolated COCs: Distinct amounts of GHR are seen in the follicular cells of the cumulus oophorus (x150). Reproduced at 74%.

Immunohistochemistry

Immunohistochemical studies in ovaries of cows and calves revealed the same distribution pattern of the GHR protein as for the mRNA (Figs. 4–6). GHR was mainly localized in the cytoplasm of oocytes of primordial and primary follicles (Fig. 4, A and C). Preincubation of specimens with GH in concentrations of 2 or 4 µg/100 µl PBS strongly reduced the signal in follicular cells and oocytes (Fig. 4B). Using GH in a concentration of 1 µg/100 µl PBS, the strength of staining in follicles was merely slightly decreased. Secondary follicles revealed the GHR protein in scattered follicle cells (Fig. 4D). In tertiary follicles, the cumulus cells showed both nuclear and cytoplasmic staining (Fig. 4E). Isolated COCs also revealed distinct amounts of GHR in the cells of the cumulus oophorus (Fig. 4F). The changes in the expression of GHR and its transcript during ontogenesis of the bovine ovary are summarized in Table 1.


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  		TABLE 1. Differential expression of GHR mRNA and protein during ontogenesis of the bovine ovary.a

Besides oocytes and granulosa cells, the germinal epithelium (Fig. 5), the endothelial cells of ovarian vessels, and the large granulosa lutein cells of corpora lutea cyclica and graviditatis were regularly labeled (Fig. 6). Whereas in calves the protein showed a localization in the ovary identical to that in cows, fetal ovaries did not display any signal in the clusters of germ cells or in the gonadal cords or follicles (Fig. 7A). Merely endothelial cells and vascular smooth muscle cells were labeled from the fourth month of pregnancy on (Fig. 7B). In contrast to observations for the mRNA (Fig. 8A), synthesis of the GHR protein did not start in primordial follicles before birth (Fig. 8B).



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  		FIG. 5. Immunohistochemical staining of GHR in the germinal epithelium of the ovary. Distinct amounts of the receptor are found in the cytoplasm of the germinal epithelial cells (x480; reproduced at 74%). FIG. 6. Immunohistochemistry of the corpus luteum: The large granulosa lutein cells are labeled for GHR (x250; reproduced at 74%). FIG. 7. Immunohistochemistry of a fetal ovary in the beginning of the fourth month of pregnancy. A) The clusters of germ cells in the ovarian cortex show no signal for GHR (x220). B) Ovarian vessels were the first to express GHR during fetal life in endothelial cells and smooth muscle cells (x200). Reproduced at 74%. FIG. 8. In situ hybridization and immunohistochemistry of a fetal ovary in the eighth month of pregnancy. A) In situ hybridization revealed distinct amounts of the GHR mRNA in the oocytes of the primordial follicles (x440). B) In contrast, the GHR protein is not synthesized in fetal primordial (arrow), primary, or secondary follicles (x420). Reproduced at 74%.

Through application of the monoclonal antibody CD68, the cells marked by the GHR antibody in the ovarian connective tissue were identified as macrophages. Labeling of these cells could not be eliminated by preincubation with recombinant GH.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we demonstrated the differential expression of the GHR and its transcript in the bovine ovary during embryonic development and folliculogenesis. In the adult, especially the oocytes of primordial and primary follicles revealed distinct amounts of GHR and its transcript. Similarly, Eckery et al. [5] localized the mRNA encoding GHR in oocytes of preantral follicles in the sheep. Strong GHR immunoreactivity has also been shown in scattered oocytes of rat ovaries [28]. The results of our in situ hybridization and immunohistochemical studies imply that the GHR plays an important role especially in the early stages of folliculogenesis. This is supported by the finding that administration of exogenous GH to cows significantly increased the number of small follicles [11]. Additionally, application of GH raised follicle diameter in in vitro-perfused rabbit ovaries in a dose-dependent manner [29].

In contrast to findings in our studies, Lucy et al. [13] could not detect the receptor protein in granulosa cells or oocytes by immunohistochemistry. They applied a monoclonal antibody directed against the extracellular domain of rat somatotropin receptor, which may recognize other epitopes than our antibody mAb 263. Using solution hybridization and Northern blotting, Lucy et al. [13] found only small amounts of mRNA encoding GHR in follicles of any size. This may be attributable to the fact that follicles were aspirated before RNA extraction. Through aspiration, the oocyte and the surrounding cells of the cumulus oophorus distinctly expressing the mRNA for GHR were removed. Also, because GHR mRNA degrades rapidly, the 1-h time before freezing of the tissue might account for difficulties in detecting GHR mRNA.

Our finding that the GHR transcript is already synthesized in primordial follicles before birth supports the concept that GHR is involved in the development and differentiation of primordial follicles both in prenatal and in postnatal life. All secondary and tertiary follicles in the fetal ovary, which degenerate after birth, expressed neither the mRNA for GHR nor the receptor protein. Thus, GH might take part in the initiation of the further development of the primordial oocytes after birth. The role of GH during this complex process remains to be evaluated.

Recently GH has been shown to accelerate nuclear maturation in bovine oocytes and to stimulate subsequent embryonic development in vitro [30]. As demonstrated by Izadyar et al. [31], this stimulatory effect of GH on oocyte maturation is dependent on the cumulus cells synthesizing the mRNA for GHR. Our in situ hybridization and immunohistochemical studies of ovaries showed that GHR and its transcript are not only synthesized in isolated cumulus cells in vitro but also in cumulus cells of the ovary during all stages of follicular development. As the GHR transcript was demonstrated in granulosa cells of the late primary follicle and secondary and tertiary follicle, GHR seems to be involved in the differentiation of the granulosa cells during the entirety of postnatal folliculogenesis. Although the cells of the cumulus oophorus synthesize distinct amounts of GHR, the protein is not secreted into the follicular fluid. In human granulosa cells, the mRNA encoding GHR has been demonstrated by RT-PCR and in situ hybridization of dominant and antral follicles [32]. The presence of functional GHRs in these cells was confirmed by competitive GH binding studies [33]. In the rat ovary, the mRNA encoding GHR was detected by Northern blot analysis of isolated granulosa cells [34]. GH has been shown to enhance cell proliferation and steroidogenesis of cultured granulosa cells in rats [6] and in cattle [8], pointing out an important role of GH in the regulation of follicular growth.

Additionally, GH may be involved in the formation of granulosa lutein cells. RT-PCR, in situ hybridization, Western blotting, and immunohistochemistry confirmed the presence of GHR and its mRNA in the large luteal cells of the bovine ovary. The strength of the signal and the localization of protein and transcript did not change in the course of pregnancy, suggesting that GHR plays a role in the differentiation of granulosa lutein cells irrespective of the fertilization process. The presence of the GHR protein in human and bovine luteal cells has already been demonstrated [13, 35]. By measuring progesterone and aromatase activity, Hutchinson et al. [36] were able to show that GH accelerated the differentiation of granulosa cells to granulosa lutein cells.

In previous studies [21], we reported that macrophages were stained by the antibody mAb 263. As in situ hybridization studies did not reveal any mRNA encoding GHR and preincubation with GH did not alter the signal in these cells, the staining of macrophages is not specific to GHR. In follicles, corpus luteum, and vessels, labeling was strongly reduced by preincubation with GH in concentrations of 2 or 4 µg/100 µl PBS. The fact that the signal was not eliminated may be due to time-dependent dissociation of recombinant GH from the receptor.

In summary, from our studies it can be concluded that the GHR and its transcript are differentially expressed in oocytes and granulosa cells of the bovine ovary during prenatal and postnatal life. Our finding that GHR is especially involved in early folliculogenesis may contribute to a better understanding of the physiological role of GH in ovarian ontogenesis and function.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. Michael Waters, University of Queensland, Australia, for kindly providing the GHR antibody. The excellent technical assistance of Mrs. Elfriede Roming and Mrs. Irene Butz is gratefully acknowledged.


   FOOTNOTES
 
1 This study was supported by the Bundesminister für Forschung und Technologie, Bonn, Germany, as a part of a larger concerted project "Fertilitätsstörungen" (01 KY 9103). Back

2 Correspondence: Sabine Kölle, Department of Veterinary Anatomy II, University of Munich, Veterinärstr. 13, 80539 Munich, Germany. FAX: 49 89 2180 2569; s.koelle{at}anat.vetmed.uni-muenchen.de Back

Accepted: May 20, 1998.

Received: December 30, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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