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A Synthetic Peptide Corresponding to Amino Acid Residues 34 to 37 of Human Follicle-Stimulating Hormone ß-Subunit Accelerates the Onset of Puberty in Male and Female Mice¹

Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York 12208

Address all correspondence and requests for reprints to: Dr. Leo E. Reichert, Jr., Department of Biochemistry and Molecular, Biology A-10, Albany Medical College, Albany, New York 12208.

	Abstract

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We have previously reported that a synthetic peptide amide corresponding to residues 34–37 (TRDL, threonine, arginine-aspartic acid-leucine) of the ß-subunit of human FSH induced prolonged vaginal estrus in normally cycling female mice (see Ref. 15). These results represented the first demonstration of an in vivo effect of a gonadotropin-related synthetic peptide on reproductive processes. We have extended these studies to examine possible effects of TRDL on the onset of puberty in female mice. In two replicated experiments, vehicle-injected control mice attained first vaginal estrus by day 39. An ip injection of 200 ug TRDL/g BW to 28-day-old prepubertal female mice, however, accelerated the onset of first vaginal estrus by 7 days in 11 of 12 (11/12) (Exp 1) and 7/9 (Exp 2) mice. Serum estradiol levels were significantly (P = 0.017) elevated in TRDL-treated mice, whereas progesterone was unchanged. Uteri of TRDL-treated mice were significantly (P = 0.003) heavier than uteri of vehicle-injected control animals of the same age and body weight. Intraluminal fluid accumulation (ballooning) at proestrus was absent in 20/21 TRDL-treated females, as were oviductal ova and ovarian corpora lutea. These phenomena are characteristic of the first estrous cycles of female mice isolated from males. To obtain further evidence for in vivo effects of TRDL, we assessed the ability of TRDL to accelerate the onset of puberty in male mice. When given as five consecutive daily ip injections of 200 ug/g BW to 28-day-old prepubertal male mice, TRDL significantly increased testis weight, when compared with vehicle-injected control mice of the same age and BW (171.3 ± 3.8 mg vs. 151.6 ± 4.3 mg, P = 0.001) and induced a 6.5-fold increase in serum testosterone levels. These studies confirm the previously reported in vivo activity of a synthetic peptide corresponding to human FSH-ß subunit 34–37 (TRDL) in adult female mice and extend its effects to the acceleration of the onset of puberty in immature male and female mice.

	Introduction

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IN MICE, elements of the social environment, as well as neural and hormonal factors, have been shown to influence estrous cyclicity, gonadotropin-induced ovulation, implantation, puberty onset, and sexual maturation (1, 2, 3, 4). Because the final stages of sexual maturation in the mouse are observed within 2–3 weeks, or can be compressed into as little as 2–3 days (depending on the olfactory environment), this animal model has proved itself an exceptionally good system for studying both the onset of puberty and the time required for the synchronization of reproductive target organ responsiveness that is associated with full fertility (5, 6, 7, 8, 9, 10).

To understand the complex interaction of factors that affect the onset of puberty in female mice, a number of laboratories have independently characterized and quantitated the timing of sexual maturation under various experimental conditions: presence or absence of adult intact males, female density, dietary protein, day length (4, 5, 11, 12). The results of these studies indicated that vaginal opening, first vaginal estrus, and increases in uterine weight can be used as reliable indices of the onset of puberty. In the male mouse, puberty is accompanied by increased steroidogenesis and testicular maturation (13).

In the present study, we have used morphological and biochemical endpoints to assess the effects of a synthetic peptide amide representing amino acid residues 34–37 (TRDL) of the human FSH (hFSH) ß-subunit on the onset of puberty in male and female mice. Our results indicate that TRDL, a peptide included within a larger receptor-binding domain of the hFSH ß-subunit, residues 33–53 (14), and shown by us to induce prolonged vaginal estrus in adult female mice (15), was also able to accelerate the onset of puberty in male and female mice. These results support the notion that development of gonadotropin-related synthetic peptides or peptide mimetics may represent a novel approach to fertility regulation and control.

	Materials and Methods

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Animal procedures
Housing.
Twenty-eight-day-old male and female Swiss Webster mice (Taconic Farms, Germantown, NY), weighing 13–16 g, were used in this study. The mice were weaned at 21 days of age and housed three per cage in 30 x 30 x 15 cm polycarbonate cages fitted with stainless steel wire lids and air filters. The cages were supported on ventilated racks (Thoren Caging Systems, Hazleton, PA) in the Albany Medical College Animal Resources Facility. Males and females were housed in separate rooms. The mice were maintained at a constant temperature (24 C) with lights on from 0600–1800 h and allowed food and water ad libitum. Access to the animal rooms was restricted to laboratory personnel. Cages and bedding (Care-Fresh, Absorption Corp., Bellingham, WA) were changed every 3 days to prevent accumulation of male or female urinary pheromones.

Peptide administration.
TRDL was dissolved in sterile PBS (pH 7.2). On day 28, female mice received 200 ug/g BW TRDL in a single ip 0.2-ml injection. Control mice received 0.2 ml PBS only. Twenty-eight-day-old male mice received five daily 0.2-ml ip injections of 200 ug/g BW TRDL. Control males were given five daily ip injections of PBS.

Vaginal perforation and first estrus.
Beginning on day 25, each female was examined daily at 0900 h for vaginal perforation. On the day of vaginal opening, and each day thereafter, vaginal lavages were taken each morning between 0900 and 1000 h to determine the day of first vaginal estrus (fully keratinized vaginal smear). Care was taken to avoid mechanical stimulation of the cervix during this procedure, to prevent pseudopregnancy. Only one attempt was made to obtain each smear. The vaginal smears were stained with 0.1% toluidine blue and staged by light microscopy. Mice were assigned to one of the four estrous stages (proestrus, estrus, metestrus, or diestrus) after nucleated vaginal epithelial cells, keratinized epithelial cells, and polymorphonuclear leukocytes were visually quantitated under a light microscope, according to the method of Allen (16).

Collection of blood, uteri, ovaries, and testes.
Between 0900 and 1000 h on the morning of proestrus or estrus in TRDL-treated females, as determined by the appearance of the vaginal smear, the mice were anesthetized with ether and exsanguinated by cardiac puncture. The blood was collected in sterile nonheparinized plastic centrifuge tubes and allowed to stand at room temperature for 1 h. The clotted blood was rimmed from the walls of the tubes with sterile wooden applicator sticks and centrifuged for 30 min at 2600 x g (Sorvall RC-3B, H-6000A rotor, Norwalk, CT). TRDL-treated and vehicle-injected control male mice were exsanguinated between 0900 and 1000 h on day 35, as described above. Serum was prepared and stored at -20 C until used.

Immediately after exsanguination, the uterus, ovaries, or testes were removed from each animal, cleaned of fat and mesentery, visually inspected for the presence of fluid (uteri), corpora lutea (ovaries) or oviductal ova (ampullae of the oviducts) under a stereomicroscope, rinsed in saline, blotted dry on filter paper, and weighed to the nearest 0.1 mg. Because of their small size, no attempt was made to separate the oviducts from the uteri. The data given, therefore, represent the combined weight of the uterus and attached oviducts. Testis weights represent the combined weight of both testes. These animal procedures were reviewed and approved by the Animal Care and Use Committee of the Albany Medical College and are in accordance with institutional guidelines.

Peptide synthesis, purification, and characterization
TRDL [hFSH-ß-(34–37)] peptide amide was synthesized in our laboratory by the solid-phase method (17) using a Rainin model PS3 automated peptide synthesizer (Rainin, Ridgefield, NJ). Fluorenylmethoxycarbonyl-protected L-amino acids were assembled on Rink (4,2',4-dimethyloxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)phenoxy resin (Advanced ChemTech, Louisville, KY). The completed peptide was cleaved from the resin with trifluoroacetic acid, using sterile deionized water, ethanedithiol, anisole, and thioanisole as scavengers. The cleaved peptide was extracted with anhydrous ether, dried by lyophilization, and purified on a Rainin Dynamax preparative HPLC column (21.4 mm x 25 cm; C18; 300-A pore diameter). The final peptide product was evaluated for purity by reverse-phase liquid chromatography on a Rainin Dynamax analytical column (4.6 mm x 25 cm; C18; 300-A pore diameter) using a linear acetonitrile gradient (0–100%) containing 0.05% trifluoroacetic acid. Fidelity of synthesis was confirmed in our laboratory by amino acid compositional analysis, and commercially by mass spectral analysis (Quality Controlled Biochemicals, Hopkinton, MA).

Estradiol and progesterone
Estradiol and progesterone levels were measured in individual serum samples in disequilibrium assays routinely used in our laboratory and described in detail previously (18, 19).

Testosterone RIA
Serum testosterone was measured in individual serum samples using a Coat-A-Count Total Testosterone kit (Diagnostics Products Corporation, Los Angeles, CA), according to the manufacturer’s protocol.

Statistical analysis
Differences in organ weight and steroid levels between TRDL-treated mice and vehicle-injected control mice were analyzed using Student’s t test and were considered significant at P < 0.05.

	Results

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Effects of TRDL on vaginal perforation and first estrus
The effects of 200 ug/g BW TRDL on vaginal perforation and first estrus are shown in Fig. 1, A and B. In two separate experiments, vaginal perforation in both vehicle-injected control and TRDL-treated mice had occurred by day 29 (Fig. 1, A and B, open bars). The timing of first vaginal estrus, however, was accelerated by TRDL treatment on day 28. In Exp 1 (Fig. 1A), completely keratinized vaginal smears (hatched bars), indicative of first estrus, were observed in 9/12 (75%) TRDL-treated mice on day 31, and in 11/12 (92%) on day 32. Only 2/6 (33%) vehicle-injected control mice had reached first vaginal estrus on either day (solid bars). After 39 days, however, all of the vehicle-injected control mice (6/6) had reached first estrus.

View larger version (29K): [in this window] [in a new window]   Figure 1. The effects of TRDL on the timing of first vaginal estrus. In two separate experiments, (A) and (B), 28-day-old female mice were given a single ip injection of 200 ug/g BW TRDL or vehicle (PBS, pH 7.2). On the day of vaginal opening, and each day thereafter, vaginal smears were obtained by lavage and examined for complete keratinization as described in Materials and Methods (Animal procedures). Each bar represents the number of animals achieving vaginal patency or first vaginal estrus. The numbers over each bar indicate the number of mice with open vaginae or in first vaginal estrus vs. the number of animals in the group.

Effects of TRDL on uterine and ovarian weight
Although TRDL treatment resulted in highly significant (P = 0.003) increases in uterine weight (Fig. 2), intraluminal fluid accumulation (ballooning) at proestrus was absent in 20/21 of the TRDL-treated mice. The uteri of vehicle-injected control mice also showed no ballooning. The uteri from TRDL-treated mice, taken at estrus, were significantly (P = 0.0035) heavier than uteri taken at the same time from vehicle-injected control mice of the same age and body weight. Oviductal ova could not be detected in the ampullae of the attached oviducts. No significant difference in ovarian weight between TRDL-treated and vehicle-injected control mice was evident (Fig. 2). When examined under a stereomicroscope, the ovaries from both groups of mice demonstrated similar morphologies. All of the follicles were approximately the same size and showed no evidence of maturation (presence of large hemorrhagic preovulatory follicles) or ovulation (presence of corpora lutea).

View larger version (14K): [in this window] [in a new window]   Figure 2. The effects of TRDL on uterine and ovarian weights. Twenty-eight-day-old female mice were given a single ip injection of 200 ug/g BW TRDL or vehicle (PBS, pH 7.2). On the morning of first vaginal estrus, as determined by the appearance of the vaginal smear, the mice were anesthetized and exsanguinated, and the uteri and ovaries were processed as described in Animal procedures. Each bar and vertical line represents the mean ± SEM weight. The number over each bar indicates the number of animals in each group. *, Uterine weight significantly (P = 0.003) heavier than vehicle-injected controls.

View larger version (13K): [in this window] [in a new window]   Figure 3. The effects of TRDL on serum estradiol and progesterone levels. Twenty-eight-day-old female mice were given a single ip injection of 200 ug/g BW TRDL or vehicle (PBS, pH 7.2). On the morning of first vaginal estrus, as determined by the appearance of the vaginal smear, the mice were anesthetized and exsanguinated as described in Animal procedures. Individual serum samples were assayed for estradiol and progesterone content. Each bar and vertical line represents the mean ± SEM estradiol or progesterone content. The number over each bar indicates the number of serum samples in each group. *, Estradiol level significantly (P = 0.017) higher than vehicle-injected controls.

View larger version (13K): [in this window] [in a new window]   Figure 4. The effects of TRDL on testes weight. Twenty-eight-day-old male mice were given 5 daily ip injections of 200 ug/g BW TRDL or vehicle (PBS, pH 7.2). On day 35, the animals were weighed, anesthetized and exsanguinated, and the testes were processed as described in Animal procedures. Each bar and vertical line represents the mean ± SEM weight. The number over each bar represents the number of animals in the group. *, Testes weight significantly (P = 0.001) heavier than vehicle-injected controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The hormonal basis of puberty in rats and mice has been well characterized. It is known that a massive and prolonged peak in serum FSH is present in female neonates of both species and that the physiological consequence of this activity is the initiation of follicular growth (5, 20). Administration of antiestrogen antibodies or antibodies to FSH arrests neonatal follicular development, suggesting that each plays an important role in this process (20, 21). By 21 days of age, the ovaries of prepubertal mice contain a full complement of follicles, and superovulation can be induced by administration of exogenous PMSG (22). An ovulatory pubertal cycle can also be rapidly initiated by exposure to male-originating stimuli (20, 21).

The efficient organization of puberty in female mice is highly dependent on the presence of male stimuli (1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 21, 22, 23). Exposure of immature females to an intact male, or to male urinary odors, accelerates sexual maturation, whereas isolation of female mice from males delays the onset of puberty. Contrary to vaginal and uterine indications that full fertility has been achieved, however, ovulation is infrequent in females housed in a male-free environment (5). In the present study, we observed similar evidences of anovulation after TRDL-induced acceleration of vaginal estrus.

The initial effect of exposure of prepubertal female mice to males is the rapid elevation of serum LH, followed by two surges in peripheral estradiol, which are not accompanied by concurrent changes in circulating FSH or progesterone (24). A similar pattern was seen in our study. Serum estradiol was increased by TRDL treatment, but progesterone levels remained unchanged. The hormonal mechanism by which male exposure accelerates puberty in immature females seems to involve this acute increase in serum LH, which acts on ovaries primed neonatally with FSH. Elevated LH stimulates the release of sufficiently high levels of estradiol, which initiate the preovulatory surge in gonadotropin secretion required for ovulation. The pivotal role of estradiol in this event has been confirmed by the observation that exogenously administered estrogen is able to mimick the effects of male exposure on the onset of puberty in immature female mice (23). Our results suggest that the effects of TRDL on the acceleration of the onset of puberty may be related to its ability to elevate endogenous estradiol in prepubertal females.

Because the hypothalamic-pituitary-ovarian axis of immature female mice is extremely sensitive to olfactory cues originating in the social environment (24), it was necessary that our experiments be carried out under the most stringently regulated conditions possible. Therefore, males and females were housed in separate and remote rooms in our Animal Resources Facility, in filtered cages supported on ventilated racks. This housing arrangement guaranteed that each cage had its own air supply and exhaust, eliminating the possibility of contamination with airborne male pheromones. We determined that changing cages and bedding every 3 days prevented excessive accumulation of female urinary pheromones, which could also influence the outcome of our studies. To assure that cross-contamination did not occur during cage and bedding changes, these tasks were performed by our own laboratory personnel, who were instructed to have two changes of clothing and shoes, one to be worn when working with females, and the other when working with males. Given these precautions, we are confident that our results represent a true estimation of the effects of TRDL on the onset of puberty in this strain of mice.

The absence of proestrus-associated uterine ballooning in TRDL-treated mice was not totally unexpected, because uterine ballooning does not usually occur in females isolated from males (5). Our data indicate that although sufficiently high levels of estradiol were present to induce vaginal cornification, the uterine response was not stimulated. These observations suggest asynchrony between the first vaginal and uterine cycles induced by TRDL treatment. Estradiol-related changes in the vaginal epithelium seemed to be out of phase with uterine changes, a phenomenon which is commonly seen in young mice ovulating for the first time (25), and in isolated females (5).

Our inability to isolate ova from the ampullae of the oviducts, or to identify corpora lutea in the ovaries of TRDL-treated mice, suggests that the first estrous cycle in these mice was anovulatory. These observations may be related to the absence of male stimuli (24) and the early age (29 days) at which TRDL induced first vaginal estrus. In a study directed at correlating the age of first estrus with ovulation, Stiff and colleagues (5) noted that females achieving first estrus before 36 days of age were typically free of oviductal ova and without corpora lutea in their ovaries. After 39 days of age, however, ova could be isolated from the oviducts of females attaining first estrus, and their ovaries contained clearly visible corpora lutea. The first estrous cycle induced by TRDL, therefore, resembles that typically seen in animals of this age housed in a male-free environment.

One commonly held explanation for the onset of puberty in the male mouse involves a shift in feedback responsivity of the hypothalamic-pituitary gonadostat that occurs at this time (13). FSH, LH, and androgen levels are known to be low and relatively stable during the first 2 to 3 weeks of life. At least 10 days before the pubertal increase in circulating androgen, a shift occurs, such that the prevailing androgen concentration is no longer sufficient to suppress FSH and LH secretion. As FSH and LH secretion increases, testicular maturation is stimulated, and the pubertal increase in androgen secretion occurs. Our studies in males indicate that TRDL administration significantly increased testis growth and elevated serum testosterone levels in 28-day-old prepubertal mice, suggesting in vivo peptide effects on testicular maturation and steroidogenesis that are consistent with the early onset of puberty in this animal model.

Although the biochemical basis underlying the in vivo effects of TRDL in immature mice is not fully understood at the present time, its ability to accelerate first estrus and elevate circulating estradiol levels in females and to stimulate testis growth and testosterone biosynthesis in males suggests mechanisms related in peptide interaction with gonadotropin receptors. We have observed TRDL to inhibit binding of both [¹²⁵I]hFSH and [¹²⁵I]hCG to bovine calf testis membranes in vitro (unpublished observations), indicating interaction with both FSH and LH/CG receptors. Thus, involvement of TRDL in LH-regulated events, i.e. testosterone biosynthesis, as well as FSH-regulated events, cannot be excluded at this time. Such involvement may be related to the similarity of TRDL to residues 42–45 (TRVL) of hCG-ß-subunit, a region reported to be important for hCG binding to LH/CG receptors (26).

The results of our study indicate that TRDL, a peptide that we have shown prolongs estrus in adult female Swiss Webster mice (15), can also accelerate the onset of puberty in both females and males of this strain. These results add further support to the concept that gonadotropin-related synthetic peptides or peptide mimetics may represent a promising approach to the development of a novel class of compounds useful in fertility control and regulation.

	Footnotes

 
¹ This work was supported by NIH Research Grant HD-13938.

Received May 13, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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