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Endocrinology Vol. 138, No. 8 3320-3329
Copyright © 1997 by The Endocrine Society

ARTICLES

A Role for Neurotransmitters in Early Follicular Development: Induction of Functional Follicle-Stimulating Hormone Receptors in Newly Formed Follicles of the Rat Ovary1

Artur Mayerhofer2, Gregory A. Dissen, Maria E. Costa and Sergio R. Ojeda

Division of Neuroscience, Oregon Regional Primate Research Center-Oregon Health Sciences University, Beaverton, Oregon 97006

Address all correspondence and requests for reprints to: Dr. Sergio Ojeda, Division of Neuroscience, Oregon Regional Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a ß-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an {alpha}-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN RATS and mice, definitive ovarian histogenesis and initiation of follicular growth are events that take place during the first week after birth (1, 2, 3, 4, 5). In the rat, follicular assembly occurs after the first 24 h of postnatal life, as part of a differentiation process that results in the massive formation of follicles during a period of less than 36 h (2, 3). Following this organizational phase, subsets of newly formed primordial follicles begin to grow and undergo another differentiation process that results in the acquisition of gonadotropin receptors and responsiveness to gonadotropins (1, 6, 7, 8, 9). A variety of physiological and biochemical approaches have established the concept that both of these key developmental events occur independently of pituitary gonadotropins (6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18).

Although it is clear that cell-cell interactions between oocytes and presumptive granulosa cells, as well as interactions between mesenchymal and pregranulosa cells, are critical for the assembly of follicles (1, 2), the cell-cell signaling molecules responsible for the organization of somatic and germ cells into follicular structures have not been identified. Though still sketchy, more is known about the factors that may control the second and third major differentiation steps in follicular development, i.e. the phase of gonadotropin-independent growth and the acquisition of gonadotropin receptors. Recently, evidence has been provided implicating two growth factors in the process regulating the growth of primary follicles, i.e. follicles with a single layer of cuboidal cells surrounding the oocyte (19, 20). One of them, the steel factor or c-kit ligand (KL) is predominantly produced in pregranulosa cells (21) and is recognized by c-kit, a receptor tyrosine kinase member of the platelet-derived growth factor family, which is predominantly expressed in oocytes (19); the other, growth differentiation factor-9 (GDF-9), a member of transforming growth factor ß superfamily, is produced only in oocytes. Both disruption of the KL gene in a natural mouse mutant (19) and experimentally induced null mutation of the GDF-9 gene (20) result in arrest of follicular growth beyond the primary one-layer stage. Neither mutation disrupted the conversion of primordial follicles (i.e. those with a single layer of flattened cells around the oocyte) into primary follicles. Thus, KL and GDF-9 control only the later phases of early follicular growth.

Several factors appear to be involved in the acquisition of gonadotropin receptors by early growing follicles. Several years ago it was shown that cAMP is a second messenger able to induce the formation of FSH receptors in granulosa cells (22) and confer the cells with responsiveness to the gonadotropin (23). The relevance of a cAMP-dependent signaling pathway to early ovarian development was suggested by the finding that feto-neonatal ovaries, which are insensitive to gonadotropins (12, 13, 14, 15, 16, 24), readily respond to cAMP with increases in aromatase activity (24), the key enzyme in estrogen biosynthesis.

More recently, the concept has been proposed that formation of FSH receptors may require the concerted action of cAMP-dependent and independent pathways (25). Activin, which is present in feto-neonatal ovaries (26), has been implicated as one of the cAMP-independent factors able to induce FSH receptors in granulosa cells in culture (27, 28). Activin may cooperate with substances operating via cAMP-dependent mechanisms to induce FSH receptor formation during early follicular growth (25). A presumed target for cAMP action in the ovary appears to be KL itself. Both oocytes and cAMP are able to induce KL synthesis in granulosa cells (21), suggesting the involvement of a cAMP-dependent signaling mechanism in the synthesis of this protein. Thus, cAMP may not only stimulate FSH receptor formation directly but also promote follicular growth via activation of c-kit/KL interactions.

But, what are the primary signaling molecules that activate cAMP formation in newly formed follicles? Because the neonatal rat ovary lacks gonadotropin receptors (7, 13, 14, 29), one has to consider the involvement of substances other than LH and/or FSH. Neurotransmitters that act via adenylate cyclase-coupled receptors and reach the ovary before the initiation of follicular growth may contribute to such a role. Vasoactive intestinal peptide (VIP) and norepinephrine (NE) are particularly attractive candidates, as both are present in the neonatal ovary (30, 31), and ligand-induced activation of their respective receptors in granulosa cells results in stimulation of cAMP formation (32, 33). In the rat ovary, both neurotransmitters act via specific receptors coupled to cAMP formation, VIP through VIP receptors type 2 (34) and NE via ß2-adrenergic receptors (35, 36). They reach the ovary via the extrinsic innervation (37, 38), which develops before the initiation of follicular growth (3), and at least one of them (VIP) has been shown to be a potent inducer of cAMP formation and aromatase activity in feto-neonatal ovaries that are unresponsive to gonadotropins (24). In the present study, we provide evidence that isoproterenol (ISO), a ß-adrenergic receptor agonist, and particularly the neuropeptide VIP, are able to activate expression of the FSH receptor (FSHR) gene in granulosa cells of newly formed follicles. This increase in FSHR gene expression is accompanied by the appearance of biologically active FSH receptors, as evidenced by ability of FSH to induce cAMP formation and stimulate follicular growth in ovaries pretreated with VIP. A partial report of these findings has appeared (39).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Pregnant Sprague-Dawley rats were purchased from B & K Universal (Fremont, CA). They were housed under controlled conditions of temperature (23–25 C) and light (14-h light, 10-h of darkness; lights on from 0500–1900 h), and given ad libitum access to food (Purina laboratory chow, Ralston-Purina, St. Louis, MO) and tap water.

Organ culture
Because the rate of follicular assembly peaks 48–72 h after birth (2, 3), the ovaries from 2-day-old rats were used to have a sizable pool of newly formed follicles. The glands were dissected under aseptic conditions, placed on sterile lens paper and cultured on metal grids at the air/medium interface in a chemically defined medium, as described (24). The medium (750 µl/well) consisted of a mixture of DMEM:F-12 media (50:50, vol:vol) containing glucose (4.5 g/liter), penicillin (100 U/ml) and streptomycin (100 µg/ml). Survival of the ovarian tissue was maximized by culturing the glands under an atmosphere of 60% oxygen, 5% CO2 and 35% nitrogen (40). The ovaries were cultured for 8–32 h depending on the parameter to be measured (see below).

Treatments
Exposure to neurotransmitters and related substances was initiated when the ovaries were placed in culture. VIP (Sigma Chemical Co., St. Louis, MO) was used at a concentration of 10 µM, a dose previously shown to be maximally effective in inducing aromatase activity and cAMP formation in feto-neonatal rat ovaries (24). The ß-adrenoreceptor agonist isoproterenol (Sigma Chemicals) was also used at 10 µM, a dose shown to be maximally effective in both stimulating progesterone release and cAMP formation in cultured granulosa cells (33, 35, 36). Secretin, a member of the VIP family that does not interact with VIP receptor type 2 (34), the VIP receptor expressed in the ovary, and does not affect aromatase activity of feto-neonatal ovaries (24), was used as a control for the VIP effects (at a 10 µM concentration). Because the rat ovary predominantly contains ß-adrenoreceptors, which are linked to both the cAMP generating system and ovarian steroidogenesis (35, 36), the {alpha}-adrenoreceptor agonist phenylephrine (10 µM) was used as a control for ISO. Purified FSH (NIH-ovine FSH-S-16, 500 ng/ml) was used to assess the presence of biologically active FSH receptors in the cultured ovaries. The diterpene forskolin (Sigma, 50 µM) was used to stimulate endogenous cAMP formation. In all experiments in which cAMP formation was assessed, the ovaries were cultured in the presence of isobutylmethylxanthine (IBMX, 0.5 mM; Sigma) to inhibit phosphodiesterase activity.

Because initial experiments, in which the ovaries were treated with test substances for up to 24 h, showed that both P-450arom and FSHR messenger RNA (mRNA) were maximally increased after a 6- to 8-h treatment, all mRNA determinations and the in situ hybridization experiments were performed after an 8-h culture period. The ability of FSH to increase cAMP formation was examined in ovaries cultured for a total of 20 h. The glands were first treated for 8 h with the test substances, washed extensively in culture medium, and exposed to FSH for an additional 12 h. When examining the effect of FSH on follicular development, the ovaries were cultured for 8 h with the test substances and then for 24 h with FSH. In all cases, one ovary was subjected to the experimental treatment and the contralateral ovary served as a control.

In situ hybridization
The ovaries were fixed by overnight immersion in 4% paraformaldehyde-borate buffer, pH 9.5, and processed for hybridization histochemistry as previously reported (41, 42). P-450arom and FSHR mRNA were detected in 10 µm cryostat sections using 35S-UTP labeled riboprobes (41, 42). Control sections were incubated with the corresponding sense RNA probes. The P-450arom cRNA was transcribed from a 484-bp cDNA corresponding to nucleotides (nt) 208–692 in the 5' end of P-450arom mRNA (43). This fragment, subcloned into the vector pBluescript KS, was obtained from a longer cDNA (15–1B) generously provided by Dr. E. Lephart (Brigham Young University, Provo, UT). For transcription, the template was linearized with SacI, and the cRNA probe was synthesized using T3 RNA polymerase. The FSHR cRNA was transcribed from a 411-bp DNA fragment (kindly provided by Dr. A. Hsueh, Stanford University, Palo Alto, CA) corresponding to nt 620-1031 in FSHR mRNA. In this case, the template was linearized with AvaII, which generates a 236 bp template. The transcription reaction was carried out using T7 RNA polymerase.

Quantitative RT-PCR (QRT-PCR)
Detailed descriptions of this method in our hands have been reported earlier (44, 45). To prepare RNA standards for the quantitation of samples, the region of FSHR mRNA between nt 630–870 was targeted for amplification. Total RNA (100 ng) from 2-day-old ovaries cultured for 8 h in the presence of forskolin, and an 18-mer polydeoxythymidine primer were used for reverse transcription. The target DNA was then PCR-amplified using a set of 18-mer oligodeoxynucleotide primers (sense primer: 5'-ACT GTG CAT TCA ACG GAA-3'; antisense primer: 5'-GCC TCC ATG AGG GTG ACA-3'). The PCR amplification consisted of 35 cycles (15 sec denaturation at 94 C, 1 min annealing at 55 C, and 2 min extension at 72 C). The PCR product was subcloned into the pGEM-T vector, and its identity was verified by sequencing. The sequencing reactions were performed using an ABI automatic DNA sequencer model 373A (Perkin Elmer, Foster City, CA) using a fluorescein dye termination reaction (Prism Ready Reaction Dye Terminator Cycle sequencing kit) and Amplitaq DNA polymerase. Thereafter, the fragment was subcloned into the SmaI site of pSP64 (polyA) (Promega Corporation, Madison, WI). As this vector contains a polyadenylated sequence adjacent to the multiple cloning site, transcription of the linearized FSHR cDNA template generates a polyadenylated sense RNA that can be subjected to RT-PCR amplification, using the same conditions employed to amplify the target FSHR gene sequence from ovarian tissue.

Ovarian RNA was extracted by the acid phenol method (46) as described (47). For the QRT-PCR procedure, 100 ng total RNA or different amounts of in vitro-synthesized polyadenylated FSHR mRNA were reverse transcribed using an 18-mer polydeoxythymidine primer and Moloney’s murine leukemia virus reverse transcriptase. Thereafter, the targeted FSHR fragment was PCR amplified using the primers described above. A 158-bp fragment of the cyclophilin gene was coamplified in each sample to control for tube "effects" and procedural variabilities (44, 45). The primers used to amplify this fragment were 18-mer oligodeoxynucleotides complementary to nt 265–282 and 405–422 in the rat cyclophilin mRNA sequence (48). The PCR reaction was carried out as reported, with some changes (35 cycles, 15 seconds denaturing at 94 C, 1 min annealing at 55 C, and 2 min extension at 72 C, ending with a 7 min extension at 72 C). The PCR products were separated on a 3% agarose gel and visualized by ethidium bromide staining. After taking a Polaroid picture, the picture was digitized using an Agfa flatbed scanner, and the signals were analyzed using an edited version of the program NIH-Image. This program yields integrated optical densities after a user-specified method of background subtraction (49). Quantitation was carried out by comparing the intensity of the signals to that generated by the FSHR mRNA standards. Values obtained were normalized according to the cyclophilin mRNA levels detected in each sample.

In some experiments, we tested the ability of forskolin to increase P-450arom mRNA levels, as a stimulatory effect of forskolin on aromatase gene expression is well documented (50, 51) and, thus, can be used as a positive control. In addition to in situ hybridization experiments for which we used the above described P-450arom cRNA, a fragment of the aromatase gene was PCR amplified from total RNA (100 ng) extracted from 2-day-old ovaries treated with forskolin for 8 h. The primers used (sense: 5'-GCA-CGA-GAA-TGG-CAT-CAT-3'; antisense: 5'-GTT-AGA-AGT-GTC-CAG-CAT-G-3') amplified the region of P-450arom mRNA comprised between nucleotides 979 and 1198. The PCR conditions used were the same described above for the amplification of an FSHR DNA fragment. As in the case of FSHR, the identity of the presumptive P-450arom PCR product was verified by sequencing.

cAMP RIA
Release of cAMP to the culture medium was determined as previously described (52). The samples and standards were acetylated before the assay to increase the sensitivity of detection (53). Under these conditions the standard curve was linear between 4 and 200 fmol/tube. The antiserum employed (rabbit anti-3',5'-cAMP-2-BSA) was purchased from ICN Biomedicals (Costa Mesa, CA) and was used at a dilution of 1:200. 125I-cAMP was purchased from Amersham (Arlington Heights, IL).

Morphological evaluation
Following treatment, the ovaries were fixed in 5% glutaraldehyde, 0.05 M cacodylic acid, pH 7.2. Thereafter, they were embedded in glycol methacrylate, serially sectioned at 2 µm, and stained with hematoxylin and Lee stain. Follicles were counted in every sixth section, and only if the nucleus of the oocyte was visible, according to a procedure described in detail elsewhere (54). Because proliferation of granulosa cells at the onset of follicular growth is not a process that occurs evenly around the oocyte, the presence of at least three rows of granulosa cells, partially or completely surrounding the oocyte, was used as the criterion to define a "growing follicle." This conservative approach considerably reduces the ambiguity of identifying growing follicles based on either the conversion of granulosa cells from flat to cuboidal or the identification of follicles with two layers of granulosa cells from those with only one layer. It is not uncommon to find follicles showing two layers of granulosa cells on one side and only one layer of semicuboidal cells on the other, a distribution that makes much more difficult the identification of truly growing follicles.

Data analysis
The paired t test was used to compare the effect of different treatments between a treated ovary and the contralateral control gland. A one-way ANOVA followed by the Fisher posthoc test was used to evaluate the statistical differences in follicle number between VIP-treated and control groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of cAMP formation increases P-450arom and FSHR mRNA levels in cultured neonatal ovaries
Cultured ovaries were treated with a single dose (50 µM) of forskolin and the changes in P-450arom and FSHR mRNA content were visualized by in situ hybridization after 6–8 or 24 h of treatment. Consistent with previous reports showing a stimulatory effect of forskolin on ovarian aromatase activity and FSHR levels (24, 25), forskolin treatment increased the steady state levels of both P-450arom and FSHR mRNA in the cultured ovaries. The change was clearly detected within 8 h of treatment (Fig. 1Go) but not at 24 h (not shown). While expression of the P-450arom gene in untreated ovaries was limited to some primary follicles located in the central portion of the gland (Fig. 1AGo; for brightfield view, see Fig. 1EGo), forskolin treatment resulted in a massive increase in P-450arom mRNA levels in many other follicles located both in the medullary and cortical portions of the ovary (Fig. 1CGo).



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  		Figure 1. Neonatal (2-day-old) rat ovaries (A and B) respond to forskolin with an increase in P-450arom mRNA (C) and FSHR mRNA levels (D), as assessed by in situ hybridization using 35S-UTP labeled cRNA probes. The ovaries were treated in vitro with forskolin (50 µM) for 8 h in a serum-free, chemically defined medium (see Materials and Methods). E and F, Brightfield views of the ovarian sections from control, untreated ovaries shown in A and B, respectively. Notice that in the untreated ovaries, detectable expression of P-450arom mRNA is limited to some primary follicles in the central portion of the ovary (A and E, arrows). Also note the much more reduced level of FSHR mRNA expression in both control and forskolin-treated ovaries (arrows in B and D, respectively, and arrows in brightfield view of B, shown in F). Bars, 100 µm.

 
FSHR mRNA expression in untreated ovaries was essentially undetectable throughout the gland, with exception of some primary follicles in the medulla that appeared to contain low levels of the mRNA (Fig. 1BGo; for brightfield view see Fig. 1FGo). As in the case of P-450arom, forskolin treatment resulted in an apparent increase in the number of follicles expressing FSHR mRNA (Fig. 1DGo). No attempts were made, however, to document this empirical observation by performing a quantitative analysis of the changes. A uniformly low hybridization signal was observed in sections incubated with either a P450arom or a FSHR sense RNA probe (not shown).

To further verify the existence of these changes, other ovaries were treated with forskolin for 6h and the changes in P450arom and FSHR mRNA levels were determined by RT-PCR. As shown in Fig. 2Go, forskolin induced a robust increase in the content of both mRNAs, thus confirming the change detected by hybridization histochemistry.



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  		Figure 2. Increase in P-450arom and FSHR mRNA levels by forskolin in 2-day-old rat ovaries as determined by RT-PCR. The ovaries were exposed in vitro to the diterpene (50 µM) for 6 h in a serum-free, chemically defined medium (see Materials and Methods). Total RNA extracted from pairs of ovaries was then subjected to reverse transcription followed by PCR amplification with primers complementary to either P-450arom (A) or FSHR (B) mRNA, as described in the Materials and Methods section. p1B15 denotes a fragment of cyclophilin mRNA coamplified with either target mRNA to correct for procedural variability. For both P-450arom and FSHR mRNAs, a single band of the expected length (220 and 241 nt, respectively) was detected. To verify the authenticity of each of these PCR products, the presumptive P-450arom and FSHR DNA fragments were isolated, cloned into the vector pGEM-T, and sequenced. F, forskolin; C, control; W, water control, no RNA input.

 
VIP and ISO increase FSHR mRNA levels in cultured neonatal ovaries
The effects of NE and VIP on ovarian steroidogenesis are mediated by receptors coupled to adenylate cyclase (ß2 and VIP-2, respectively, see above). To determine if activation of these receptors can reproduce the stimulatory effect of forskolin on ovarian FSHR gene expression, 2-day-old ovaries were exposed to ISO (to stimulate ß-adrenergic receptors) or VIP for 8 h, and the resulting mRNA levels were assessed by QRT-PCR. In each assay, different amounts of in vitro transcribed FSHR mRNA were reverse transcribed and PCR amplified to generate standard curves for quantitation of the experimental samples. Ethidium bromide staining of the DNA products showed that increasing amounts of this in vitro-transcribed FSHR mRNA are amplified in a linear fashion (Fig. 3AGo). Densitometric analysis yielded standard curves (example shown in Fig. 3BGo) used to quantitate the changes in ovarian FSHR mRNA induced by the ISO and VIP treatments. Both ISO and VIP increased FSHR mRNA levels (example shown in Fig. 3CGo). Quantitation of the changes detected in three different experiments demonstrated that ISO induced a 2-fold increase in FSHR mRNA (P < 0.05), and VIP induced a 3-fold increase (P < 0.025) (Fig. 3DGo).



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  		Figure 3. Ability of ISO and VIP (10 µM each) to increase the steady-state levels of FSHR mRNA in 2-day-old neonatal rat ovaries, as measured by QRT-PCR. The glands were exposed in vitro for 8 h to either ligand. One of the ovaries from each animal was treated with the test substance, and the contralateral ovary served as a control. A, Ethidium bromide staining of the standard curve generated by RT-PCR amplification of various amounts of an in vitro synthesized polyadenylated FSHR mRNA fragment corresponding to the identical cellular mRNA sequence targeted for amplification. B, Regression analysis of the standard curve shown in A. C, Results of one experiment in which the ovaries were treated with either ISO or VIP. p1B15 refers to a DNA fragment of cyclophilin (a constitutively expressed gene) coamplified with the target FSHR mRNA to correct the estimated FSHR mRNA values for procedural variability. D, Calculated FSHR mRNA values derived from three experiments (each with three to four pairs of ovaries), including the experiment shown in C. Bars represent mean ± SEM.Numbers inside bars are number of ovaries per group. Co, Untreated controls; ISO, isoproterenol; VIP, vasoactive intestinal peptide. *, P < 0.05; **, P < 0.025 vs. Co.

 
VIP and ISO induce the appearance of biologically active FSHR in neonatal ovaries
To determine if the increase in FSHR mRNA content elicited by VIP and ISO was accompanied by a corresponding change in functional FSHR molecules, the ovaries were first exposed to VIP or ISO for 8 h and then to FSH (in the absence of either "priming" agent) for 12 h. At this time, the culture medium was collected for cAMP measurement. As shown in Fig. 4Go, FSH was effective in promoting cAMP formation only when the ovaries were pretreated with either VIP or ISO. The "priming" effect of ISO (A) was less prominent than that of VIP, but VIP also appeared to have a strong residual effect (B). Ovaries treated with VIP showed significantly elevated cAMP levels 12 h after discontinuation of the treatment, in the absence of FSH stimulation. Despite this residual effect, treatment of VIP-primed ovaries with FSH resulted in a further increase in cAMP levels (Fig. 4BGo). The effects of ISO and VIP appeared to be specific, as they were not reproduced by phenylephrine, an {alpha}-adrenergic agonist (Fig. 4AGo), or secretin (Fig. 4BGo), a member of the VIP family that is not recognized by VIP receptors type 2, the predominant receptor subtype expressed in the ovary (34).



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  		Figure 4. ISO and VIP induce the appearance of functional FSH receptors in neonatal 2-day-old rat ovaries, as determined by the ability of FSH to stimulate cAMP formation. Following an 8-h exposure to either ISO or VIP (10 µM each), the ovaries were cultured in the presence of FSH (500 ng/ml) for 12 h and cAMP released to the culture medium was measured by RIA at the end of this period. Bars are mean ± SEM. Numbers inside bars are number of ovaries per group. A, **, P < 0.01 vs. all other groups, regardless of FSH treatment. B, **, P < 0.01 vs. control (Co) group not treated with FSH; *, P < 0.025 vs. both control and secretin (Secr)-primed groups treated with FSH, and vs. VIP-primed group not treated with FSH. Phe, Phenylephrine.

 
To determine if the increase in FSHR coupled to the cAMP generating system is linked to end-stage physiological events, other ovaries were sequentially exposed to VIP and FSH and processed for morphological examination. Figure 5Go depicts representative photomicrographs of an ovary treated with VIP alone and no FSH (A), an FSH-treated ovary not preexposed to VIP (B), and an ovary pretreated with VIP before exposure to FSH (C). It is apparent that while ovaries treated with FSH alone show some follicles with three or more granulosa cell layers, the number of growing follicles is noticeably increased by treating the ovaries with VIP before exposing them to FSH. The quantitative analysis of these data, shown in Fig. 6Go, demonstrates that FSH alone modestly increased the number of "growing" follicles (i.e. follicles showing three or more granulosa cell layers) when compared with control ovaries (Co,-FSH) not exposed to the gonadotropin (P < 0.05). Preexposure to VIP, however, significantly (P < 0.05) enhanced the FSH effect.



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  		Figure 5. A, Microphotograph of an ovary pretreated with VIP for 8 h and then cultured for 24 h in the absence of FSH. Notice that most follicles are either primordial or primary, but not growing, i.e. with three or more layers of granulosa cells. B, Microphotograph of an ovary exposed to FSH for 24 h without pretreatment with VIP. Notice the appearance of some follicles with three layers of granulosa cells (arrow). C, Example of an ovary pretreated with VIP for 8 h and then exposed to FSH for 24 h. The presence of several follicles with three layers of granulosa cells (arrows) is readily apparent. Bars, 24 µm.

 


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  		Figure 6. VIP induces the appearance of FSH receptors able to initiate follicular growth in neonatal 2-day-old rat ovaries in vitro. The ovaries were exposed to VIP (10 µM) for 8 h and then to FSH (500 ng/ml) for 24 h. The number of "growing" follicles, i.e. those showing three or more layers of granulosa cells were counted on serial 2-µm sections. Bars are mean ± SEM. Numbers inside bars are number of ovaries per group. Co, Control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study suggest that neurotransmitters that affect ovarian function via activation of membrane anchored receptors linked to the cAMP generating system are involved in the cytodifferentiation process by which ovarian follicles acquire responsiveness to gonadotropins.

The pivotal role of cAMP in inducing granulosa cell differentiation is now well established. cAMP not only induces the synthesis of FSH receptors (22) but also mediates the subsequent inductive effect of FSH on LH receptors (55). Both the mechanisms leading to cAMP formation and the downstream signaling pathways mediating the effects of the nucleotide on ovarian function are already in place before the formation of the first primordial follicles. For instance, formation of cAMP can be enhanced in fetal ovaries by direct activation of the guanine nucleotide protein-adenylate cyclase complex with forskolin (24), cholera toxin (9), or prostaglandins (12, 13, 56). In turn, a rise in cAMP levels results in the activation of at least two different processes in presumptive granulosa cells. On the one hand, cAMP increases the ability of the cells to produce estrogen by inducing aromatase activity (24); on the other, it increases expression of the gene encoding the steel factor (21), a polypeptide produced by pregranulosa cells and thought to be one of the cell-cell signaling molecules involved in the growth of primary follicles beyond the one-layer stage (19). Thus, cAMP may regulate follicular development by inducing the synthesis of gonadotropin receptors, stimulating the synthesis of key steroidogenic enzymes, and enhancing the synthesis of cell-cell signaling molecules involved in oocyte-granulosa cell communication.

Although these observations suggest an important role for cAMP in early follicular growth, little if anything is known about the first messengers responsible for the inductive increase in cAMP formation that presumably precedes the appearance of FSH receptors. In addition to gonadotropins themselves, neurotransmitters acting via seven-transmembrane-domain receptors linked to adenylate cyclase are probably the most conspicuous group of first messengers linked to the cAMP generation system known to affect ovarian function. Because two of these transmitters, NE and VIP, are already present in the ovary at the time of folliculogenesis (30, 31), before the newly formed follicles acquire FSH receptors (57), the possibility exists that the initial increase in cAMP formation leading to the formation of these receptors is, at least in part, a neurotransmitter-dependent event.

Our results show that ISO, a ß-adrenergic receptor agonist, and especially VIP, increase FSHR gene expression and induce the formation of biologically competent FSH receptors in neonatal rat ovaries. The lower potency of ISO when compared with VIP may be due to several factors, including a relative paucity of ß-adrenoreceptors, uncoupling of the receptors to the cAMP generating system, and degradation of the agonist by the cultured tissue. The effectiveness of VIP, on the other hand, is in keeping with previous observations demonstrating the potency of the peptide in stimulating cAMP formation and inducing aromatase activity in feto-neonatal ovaries (24). The finding that exposure of these young ovaries to VIP results in 1/8th of the cAMP levels induced by forskolin, but in comparable levels of aromatase activity (24), strongly suggest that VIP, rather than having a general effect, targets a circumscribed subpopulation of ovarian cells.

Because the ovary seems to only express VIP2 receptors (34), it is likely that these are the receptors activated by the peptide in neonatal ovaries. VIP2 receptors, however, can be activated with equal potency by other members of the VIP family, such as helodermin (58) and pituitary adenylate cyclase activating peptides (PACAPs) (34, 58), raising the possibility that VIP is not the only (or more potent) neuropeptide interacting with VIP2 receptors able to induce FSHR expression in early follicles. The demonstration that PACAP coexists with VIP in nerve fibers innervating the human ovary (59), supports this notion.

Hirshfield et al. (60) recently demonstrated that the first follicles to start growing are those assembled near the ovarian hilum. Because this is also the first region of the rat ovary to be innervated during feto-neonatal life (3), it appears reasonable to assume that newly formed follicles in this region of the ovary are immediately exposed to neurotransmitters released from the invading nerve fibers. Thus, there seems to be both a temporal and a spatial opportunity for neurotransmitters acting via adenylate cyclase-coupled receptors to influence early follicular development, via induction of FSHR synthesis.

The critical importance of the process by which follicles become responsive to gonadotropins must require a control system endowed with both interactive and redundant regulatory loops. The demonstration that activin is a potent inducer of FSHR in granulosa cells (27, 28), and that its effect is synergistic to that of cAMP (25), has led to the suggestion that activin works cooperatively with cAMP in inducing differentiation of primordial granulosa cells (25). It now appears that activin is not the only member of the transforming growth factor-ß (TGFß) superfamily that may be involved in this process. TGFß2 is also a candidate for this role because it is expressed in pregranulosa cells closely apposed to primordial follicles (61) and effectively increases FSHR mRNA levels in cultured granulosa cells (62). It does not appear that acquisition of FSHR is the only process required for subsequent gonadotropin-dependent follicular growth. Null mutation of the GDF-9 gene, which prevents the growth of follicles beyond the one-layer cuboidal cell stage, does not affect FSHR gene expression, suggesting the need of additional, GDF-9-dependent events for the completion of the cytodifferentiation process that leads to gonadotropin dependency.

Thus, early follicular growth may be envisioned as a multiphased process regulated by factors acting at different levels along the developmental pathway. The ability of primordial follicles to grow in vitro to the primary stage in the absence of extragonadal factors (17, 18) indicates that ovarian nerves are not required for the onset of follicular growth. While the factors controlling this process remain unknown, subsequent growth of primary follicles appears to depend on oocyte-derived signaling molecules, such as GDF-9 (20), and proteins produced by granulosa cells, such as the c-kit ligand (19). Our results, considered in conjunction with the data recently reported by other laboratories (25, 62) suggest that the subsequent cytodifferentiation process that leads to the acquisition of FSH receptors by granulosa cells is not only regulated by locally produced growth factors acting via cAMP-independent signaling pathways (such as activin and TGFß2), but it may also be facilitated by neurotransmitter ligands acting via the cAMP generating system (such as NE and VIP). Within the context of normal in situ development, it may be contended that primordial follicles exposed to these neurotransmitters might have a developmental advantage over those located beyond the area to which NE/VIP may diffuse upon release from nerve endings. By stimulating cAMP formation, and inducing FSHR synthesis, NE and/or VIP may contribute to selecting discrete subsets of follicles for gonadotropin dependency. Consistent with this interpretation, neonatal sympathectomy brought about by the immunoneutralization of nerve growth factor has been shown to result in stunted follicular development, reduced steroidogenesis, and delayed puberty (63).


   Acknowledgments
 
We thank Ms. Janie Gliessman for typing the manuscript and Ms. Diane Hill for editorial assistance.


   Footnotes
 
1 Supported by NIH Grant HD-24870 (SRO), P-30 Population Center Grant HD-18185, and RR-00163 for the operation of the ORPRC. Back

2 Visiting professor at the ORPRC supported by a Heisenberg fellowship (MA 1080/4–1) from the Deutsche Forschungsgemeinschaft (Germany). Present address: Anatomical Institute, Technical University, D-80802 Munich, Germany. Back

Received February 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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