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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4513-4520
Copyright © 2004 by The Endocrine Society

Contraceptive Action of a Gonadotropin-Releasing Hormone II Analog in the Rhesus Monkey

Theresa M. Siler-Khodr, Fu-Qing Yu, Peng Wei, Shi-Xin Tao and Yi-Xun Liu

Center for Investigation of Cell Regulation and Replication (T.M.S.-K.), San Antonio, Texas 78229; and The State Key Laboratory of Reproductive Biology (F.-Q.Y., P.W., S.-X.T., Y.-X.L.), Institute of Zoology, Beijing 100080, China

Address all correspondence and requests for reprints to: Theresa M. Siler-Khodr, Ph.D., Center for Investigation of Cell Regulation and Replication, 7711 Louis Pasteur, Suite 509, San Antonio, Texas 78229. E-mail: swgenetics{at}aol.com; or Yi-Xun Liu, Ph.D., The State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. E-mail: liuyx{at}panda.ioz.ac.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH I (mammalian GnRH) was previously thought to be the only isoform of GnRH expressed in mammals, but GnRH II (chicken II GnRH) has now been identified in tissues of numerous mammalian species. Specific high-affinity receptors, which bind GnRH II and its analogs, have been identified throughout the reproductive tract, and GnRH II regulation of progesterone and human chorionic gonadotropin have been demonstrated. Thus, we hypothesized that GnRH II acts as a paracrine factor to regulate extrahypothalamic tissue functions and could be used as an effective contraceptive agent.

In these studies, we examined the effect of a stable analog of GnRH II (GnRH II analog) on ovarian steroidogenesis, implantation, and maintenance of pregnancy in the rhesus monkey. GnRH II analog or saline was administered by osmotic minipumps on d 1–6, d 6–11, or d 11–17 to cycling monkeys mated with fertile males. Circulating progesterone and estradiol were determined during the luteal phase, and the cycle length before, during, and after treatment was observed. Circulating monkey chorionic gonadotropin was used to determine implantation. In animals treated with GnRH II analog on d 1–6, no pregnancies resulted; but in saline-treated controls, five of eight animals (62.5%) became pregnant. In animals treated with analog on d 6–11, two of five (40.0%); and on d 11–17, one of three (33.3%); implanted and normal pregnancies ensued. Cycle length or progesterone production was not affected by analog treatment.

These data demonstrate that this GnRH II analog can act as a contraceptive agent when administered chronically around the time of ovulation or early luteal development. These findings suggest that GnRH II may play a role in reproductive physiology and that GnRH II analogs may serve as an effective, nonsteroidal, postcoital contraceptive.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GONADOTROPIN-RELEASING HORMONE I, a decapeptide known to regulate pituitary LH and FSH, was first identified in the hypothalamus 3 decades ago (1). It was thought to be the only isoform of GnRH expressed in mammals. Multiple forms of GnRH were not recognized to exist in mammalian species until GnRH II (originally named chicken II GnRH) was demonstrated in the brain of the tree and musk shrew (2, 3, 4). The expression of the mRNA for GnRH II in the primate brain (5) and in the human (6, 7) has recently been reported. However, GnRH II has less than one tenth or one third the LH-releasing activity of GnRH I in rat and sheep pituitaries in vitro (8, 9), and we have observed no effect of GnRH II analog on baboon pituitary LH release (10). On the other hand, Lescheid et al. (5) and Densmore and Urbanski (11) have reported an increase of LH after GnRH II when administered systemically during the luteal phase to rhesus monkeys, having little effect on LH in the follicular phase. These data have led us to question the role of GnRH II in the regulation of pituitary LH release and to consider alternative functions for GnRH II.

The ovary and the placenta are also known to produce GnRH-like peptides (12, 13, 14). Numerous reports have described GnRH-human chorionic gonadotropin (hCG)-steroidal-prostaglandin axis in chorionic tissues (15). The presence of a GnRH receptor was first described in rat luteal cells in 1979 (16). A GnRH receptor in human corpus luteum was later described by Bramley et al. (17), and the expression of an mRNA for GnRH I in human ovarian tissues was described (18, 19). However, the affinity of the ovarian and placental receptor for GnRH I or its analogs is greatly reduced compared with that in the pituitary (17). Other investigators have described GnRH I mRNA expression in the fallopian tube (20), the early embryo (21), and the endometrium (22, 23). Most studies describing the activity of GnRH I and its analogs in extrapituitary tissues have been plagued with the finding of low-affinity binding sites and the high concentration of GnRH I required to affect tissue function.

These data led us to investigate the possibility that other GnRH isoforms are active in reproductive tissues, having higher binding affinity and enhanced bioactivity at these sites. Our findings of GnRH II production, its receptor binding, and activity in the human placenta (24) and the baboon ovary (10) support this hypothesis. The expression of mRNA for GnRH II from human granulosa cells in vitro (25) and in the human endometrium (26) have been reported. Our data indicate the presence of two GnRH receptors, and the potent activity of GnRH II and its analogs, via specific receptors, on the inhibition of progesterone production in ovary and hCG release in the placenta led us to propose that GnRH II may regulate reproductive tissues functions related to ovulation, fertilization, transport, implantation, and pregnancy in these tissues, and that its chronic administration may exert a contraceptive activity.

We designed a GnRH II analog, which is stable in the presence of circulating, ovarian, endometrial, and chorionic peptidases (27, 28). This GnRH II analog avidly binds the receptor in the ovary and placenta and regulates ovarian progesterone, uterine prostaglandins, and chorionic hCG production (10, 24). To test our hypothesis, we have administered this analog at different times during the luteal phase to cycling rhesus monkeys mated with normal males. The action of this analog on the circulating levels of progesterone, estradiol, and monkey CG (mCG) during the luteal phase, the subsequent cycle, and/or pregnancy was determined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal procedures

Rhesus monkeys (Macaca mulatta) were used in these studies. The experiments were done in Xinye Primate Breeding Center in Henan province, China. The protocol was approved by the Academic Committee for Animal Experimentation of the Institute of Zoology of Chinese Academy of Sciences. Twenty-four female monkeys of 5–7 yr of age, which were regularly cycling (as determined from at least two recorded cyclic bleeds), all having a previous normal term pregnancy, were used in the experiment. The monkeys were housed in individual cages and fed with pelleted feed in the morning and evening and vegetables at noon. The time of ovulation was estimated from the length of the prior two menstrual cycles. Retrospectively, the time of ovulation in the treatment cycle was confirmed using the LH and/or estradiol peak, progesterone concentrations, the next menses, and/or the subsequent cycle’s hormonal patterns. On the day before the estimated time of ovulation (d –1), the female monkey was introduced into a cage with a reproductive adult male monkey for mating, and removed on the third day (d 1), i.e. mating was for two complete days. The males were chosen from a group of fifteen sexually active males with established fertility and documented normal sperm counts. The activity of GnRH II at various times of the luteal phase was studied by delivery of GnRH II analog, D-Arg(6)-GnRH II-aza-Gly(10)-NH2 synthesized by Sigma-Gynosys (Woodlands, TX), via primed osmotic minipump (Durect Corp, Cupertino, CA). Primed minipumps were implanted sc in the upper-back under light ketamine anesthesia. Primed pumps were loaded with either saline or analog in saline, as described below, and were implanted on d 1 (post ovulation), d 6, or d 11, respectively. Vials containing dried aliquots of GnRH II analog or saline were blindly resuspended with sterile saline to make a concentration of 66.7 µg GnRH II analog/ml or 0 µg/ml, respectively. The minipumps were loaded with GnRH II analog or with saline solution and allowed to prime overnight (16 h) in sterile PBS, such that the implanted pumps would deliver analog at approximately16 µg/d or saline for 6 d. On d 1 (post ovulation), d 6, or d 11, monkeys were implanted with the preequilibrated pumps after a basal blood sample was collected. The analog and saline pumps were blindly implanted, contemporaneously and at random, for each time of the luteal phase studied. The pumps were left in place for 18 d. Blood samples were then collected every other day for the next 18 d. Thereafter, the blood samples were drawn every week until menses or pregnancy was confirmed. The samples were processed as plasma-EDTA (4 mM/ml blood)-Bacitracin (50 U/ml blood) and frozen at –20 C until used for hormone assays as described below. During and after the treatment, the monkeys’ activities, including the cycles and pregnancies, were monitored. A total of 24 monkeys were studied: eight animals were implanted on d 1 (five with analog and three controls), nine on d 6 (five with analog and four controls), and seven around d 11 (three with analog and four controls).

Hormonal analyses

Progesterone. Progesterone was measured in monkey plasma using the Diagnostic Products Corporation (DPC, Los Angeles, CA) immunoradiometric Coat-a-Count assay for progesterone. Standard and samples (50 µl in duplicate) were analyzed according to the described procedure and counted using a Packard COBRA {gamma}-counter to 2% efficiency. The data were calculated using the COBRA software and results are expressed as nanograms per milliliter. The sensitivity of the assay was 0.2 ng/ml, and the coefficient of variation within and between assays was 3.9 and 6.8%, respectively.

Estradiol. Estradiol was measured in the monkey plasma using the DPC’s immunoradiometric Coat-a-Count procedure for estradiol. Standard and samples (100 µl in duplicate) were analyzed according to the described procedure and counted using a Packard COBRA {gamma}-counter to 2% efficiency. The data were calculated using the COBRA software, and results are expressed as picograms per milliliter. The sensitivity of the assay was 20 pg/ml, and the coefficient of variation within and between assays was 4.1 and 9.2%, respectively.

LH. Using a rabbit polyclonal antibody to cynomolgus monkey LH (AFP342994) at a final dilution of 1/1,000,000, a specific and sensitive RIA for rhesus LH was implemented. The reagents were provided by Dr. Alfred Parlow through the National Pituitary Agency. The standard was recombinant cynomolgus monkey LH (rec-moLH-RP-1, AFP-6936A). Highly purified recombinant cynomolgus monkey LH (AFP-6936A) was radioiodinated by the method of Hunter and Greenwood (29). After a 3-d incubation of sample or standard with antibody at 4, labeled monkey LH was added (20 pg/tube) and the incubation continued for 3 d at 4 C. The bound hormone was precipitated using magnetic beads coated with anti-rabbit {gamma}-globulin (Qiagen, Inc., Valencia, CA). Assay sensitivity was 8 ng/tube or 80 ng/ml, and the intra- and interassay coefficients of variation were 6.0 and 10.2%, respectively.

mCG. A specific and sensitive RIA for mCG was developed using reagents provided by Dr. Gary Hodgen. The procedures were as previously described (30) using a rabbit antiserum to the ß-subunit of ovine LH (1/100,000 final concentration). 125I -ovine LH was used as label (50 pg/tube), and the standard was mCG-RP-2. Incubation of standard or samples with antiserum was for 1 d at 4 C. Label was added and incubation continued for 1 d at 4 C. Precipitation of bound hormone was effected using second antibody-conjugated magnets. Assay sensitivity was 10 ng/ml, and the coefficient of variation within and between assays was 7.2 and 11.1%, respectively.

GnRH II analog. A specific and sensitive RIA for GnRH II analog was developed using a rabbit antibody generated by Sigma-Gynosys to keyhole limpet hemacyanin-conjugated GnRH II analog. The procedure was as previously described (24). Standard was GnRH II analog. Incubation of standard or samples with antiserum was for 2 d at 4 C. Label was added and incubation continued for 2 d at 4 C. Precipitation of bound hormone was performed using second antibody-conjugated magnets (Qiagen, Inc.). Assay sensitivity was 0.060 nM, and the coefficient of variation within and between assays was 10.0 and 11.0%, respectively.

Statistical analyses

Luteal progesterone concentrations for the control animals were calculated as the daily mean of the running average for the prior day and that day, starting from the first day post ovulation. The first day post ovulation was retrospectively established using hormonal and menstrual pattern data from each saline-treated animal and their subsequent cycle. This allowed for the comparison of all saline-treated animals with analog-treated animals at each luteal phase studied. Luteal progesterone patterns were compared among control animals and those treated with GnRH II analog at each of the three times of the luteal phase using two-way ANOVA. Bonferroni’s test was used to determine points of significant variance for a particular treatment. P value < 0.05 was considered significantly different. Cycle length, luteinization, and pregnancy rates were compared in saline controls and the three different GnRH II analog-treated groups using Fischer’s exact test. P value < 0.05 was considered significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animal cyclicity and pregnancy

The time of ovulation (d 0) in each animal was estimated from the previous two cycle lengths and was used to determine the day of treatment. Of the 11 control animals implanted with saline, one died of diarrhea and thus was removed from the analyses. Two animals were not exposed to the male at the time of ovulation, as determined by the LH and/or estradiol peak, and thus were not included in the pregnancy analysis (Table 1Go). All thirteen GnRH II analog-treated monkeys were exposed to males during their fertile period.


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  		TABLE 1. Effect of saline or GnRH II analog on luteal length and implantation

 
In the saline-treated cycles, luteinization occurred in eight of the 10 control animals (Table 2Go). Of the eight controls appropriately mated with males, six had luteinized cycles, and five implanted (62.5%), i.e. five of the six luteinized animals became pregnant (83.3%). One pregnancy was lost, and the other four had normal term deliveries and outcomes (Tables 1Go and 2Go). Of the five animals treated with GnRH II analog on d 1–6, there was evidence of luteinization in two of the animals, yet none became pregnant (0%). Of the five animals treated with this GnRH II analog on d 6–11, luteinization was observed at the time of treatment in three of the animals, and two of the animals implanted and delivered normal babies (40%). Of the three animals treated on d 11–16, luteinization was ascertained in one animal that implanted and continued a normal pregnancy (33%). The overall pregnancy rate for the analog-treated animals was 23% vs. 62.5% for saline-treated controls.


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  		TABLE 2. Comparison of pregnancy rate in saline- vs. GnRH II analog-treated monkey during different times of the luteal phase

 
Circulating hormonal concentrations in control animals

Circulating progesterone in the saline-treated luteinized controls is shown in Fig. 1Go. Maximal progesterone was attained on d 7–9 and subsequently declined. The five animals that became pregnant were not included in the data analysis from d 13 because implantation was confirmed by increasing mCG (greater than 20 ng/ml). In four saline controls that became pregnant and continued to term, mCG increased, attaining peak levels of greater than 2000 ng/ml around d 16–29, falling to 30 ng/ml by 35 d post ovulation, and falling to less than 20 ng/ml by 42 d post ovulation. Maternal circulating progesterone and estradiol concentrations were sustained. In the one saline-treated animal that had an implanted pregnancy but did not continue to term, maximal mCG levels of only 313 ng/ml were attained on d 27, then fell to nonpregnancy levels by d 36 post ovulation, as did progesterone concentrations.



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  		FIG. 1. Maternal circulating progesterone (mean ± SEM) for the eight saline-treated animals with evidence of adequate luteinization in the treated cycle is shown for each day post ovulation.

 
Effect of GnRH II analog treatment on d 1–6 post ovulation

In the five animals treated on d 1–6 of the luteal phase, the circulating GnRH II analog concentration was 0.157 ± 0.040 nM (mean ± SEM) by d 3. None of the five animals became pregnant, although luteinization was already apparent in two of the animals. The number of pregnancies in these GnRH II analog-treated animals on d 1–6 was significantly less than that observed in controls (P < 0.0083). The progesterone concentrations in the two analog-treated animals, which were luteinized at the time of treatment, initially increased and then were similar to the luteinized controls on d 4–16 post ovulation (Fig. 2Go). In the nonluteinized animals, progesterone did not increase with GnRH II analog treatment. The mean progesterone for animals treated on d 1–6 did not differ from the circulating progesterone in the saline controls (Fig. 3Go). Estradiol was not different in these animals, compared with the saline-treated animals. Cycle length was not significantly effected in the GnRH II analog-treated animals, having a mean ± SEM of 13.8 ± 1.7 d in analog-treated animals as compared with 15.8 ± 1.6 for saline-treated animals.



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  		FIG. 2. Maternal circulating progesterone for each of the five d 1–6 GnRH II analog-treated animals is compared with the circulating progesterone (mean ± SD) in saline-treated controls. Lut, Luteinized; Preg, pregnant.

 


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  		FIG. 3. The maternal circulating progesterone animals (mean ± SEM) for all saline-treated animals (n = 10) is compared with that for GnRH II analog-treated animals (mean ± SEM) on d 1–6 (n = 5), d 6–11 (n = 5), and d 11–17 (n = 3).

 
Effect of GnRH II analog treatment on d 6–11 post ovulation

In the animals treated with GnRH II analog beginning on d 6, the maximal GnRH II analog attained peak levels on d 10 (0.171 ± 0.005 nM, mean ± SEM). Of the five treated animals, three were luteinized, having similar progesterone concentration before and during treatment as that of luteinized controls (Fig. 4Go). Two of these animals became pregnant, and normal newborns were delivered. Mean progesterone for the animals treated on d 6–11 were not different from controls (Fig. 3Go). The cycle length (mean ± SEM) in the three animals that were not pregnant was not different from controls (15.3 ± 2.3 d).



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  		FIG. 4. Maternal circulating progesterone for each of the five d 6–11 GnRH II analog-treated animals is compared with the circulating progesterone (mean ± SD) in saline-treated controls.

 
Effect of GnRH II analog treatment on d 11–16 post ovulation

Of the three animals treated with GnRH II analog on d 11–16, maximal analog concentration was attained on d 13 (0.197 ± 0.026 nM, mean ± SEM). One had adequate luteinization as ascertained by progesterone levels (Fig. 5Go). This animal was pregnant, and normal hormonal production and a normal pregnancy outcome were observed. In the other two animals, luteinization could not be ascertained, but at this time of the luteal phase, increased progesterone production could have passed. These two analog-treated animals did not become pregnant. The mean progesterone for the analog-treated animals was similar to that for control animals (Fig. 3Go). In the two nonpregnant animals, menses occurred on d 12 and 21, which did not vary significantly from the saline-treated controls.



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  		FIG. 5. Maternal circulating progesterone for each of the five d 11–17 GnRH II analog-treated animals is compared with the circulating progesterone (mean ± SD) in saline-treated controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The existence of multiple forms of GnRH in nonmammalian species has been recognized for decades (31, 32), but only recently have multiple isoforms of GnRH in mammals been considered. Our findings of GnRH II production and high-affinity specific binding sites for GnRH II in the baboon ovary (10) and human placenta (24) led us to hypothesize that GnRH II may have potent paracrine activity in extrahypothalamic tissues, such as the ovary, ovum, sperm, fallopian tube, endometrium and placenta. Our demonstration that GnRH II has potent bioactivity in reproductive tissues with limited affect on pituitary function led us to propose that GnRH II might be used as a site-specific contraceptive. These studies demonstrate a contraceptive activity for this GnRH II analog in the normal cycling rhesus monkey. The contraceptive action of the GnRH II analog was effected around the time of ovulation into the very early luteal phase of the cycle, when administered over this 6-d period. Treatment with this GnRH II analog in the periovulatory-early luteal phase resulted in a total inhibition of implantation, without inhibition of luteinization or alteration of cycle length. However, to determine the minimal length of treatment to effect this activity at this phase of the cycle, further investigation will be needed. Treatment on d 6–11 or d 11–17 using this concentration GnRH II analog did not significantly inhibit implantation or continuing pregnancy and was without significant effect on progesterone production or cycle length. Although, it is possible that an effect of the analog might be observed using a larger number of animals or a higher dose of analog during the mid- or late luteal phase.

The mechanism of the contraceptive activity of GnRH II analog during the periovulatory-early luteal phase might be multifactoral. Inhibition of some function of the corpus luteum, the ovulated ovum, the fertilizing sperm, the transport of the egg through the fallopian tube, and/or priming of the endometrium are all possible sites of action. Because the contraceptive activity was observed during the periovulatory-early luteal phase, these findings suggest that this GnRH II analog may act on any or all of these sites to disrupt ovulation, fertilization, and/or implantation. Prior studies have demonstrated the presence of GnRH-like activity and its receptors in any or all of these tissues and thus support the hypothesis that this analog may act at these sites.

The production of GnRH II and its action on human (10) and primate (25) ovarian tissues have been demonstrated. In our studies using fresh granulosa and ovarian cell cultures and in the study by Kang et al. using long-term granulosa cells in vitro, an inhibition of progesterone production was observed with GnRH II analog treatment. The expression of GnRH II mRNAs is regulated by estradiol and progesterone, with progesterone having an inhibitory activity on GnRH II (33). Differential regulation and activity of these two GnRH isoforms suggests that independent regulatory mechanisms exist. In addition, we have demonstrated two different GnRH receptor activities in baboon ovarian tissue, one of which has high affinity for GnRH II. In the present studies, an inhibition of circulating progesterone was not observed using this dose of GnRH II analog at any time of the luteal phase, suggesting that this may not be the mechanism of the contraceptive action effected in these animals. It should be noted that the maximal circulating concentration of GnRH II analog attained in any of the treated animals with this protocol was 0.20 nM, i.e. less than the effective concentration needed to inhibit progesterone production from granulosa cells in vitro.

Other tissues may be affected by this GnRH II analog, leading to a contraceptive action during fertilization and/or transport of the fertilized ovum. The human and monkey ova have been shown to contain GnRH I-like activity (21). An action on the fertilizing sperm might be considered as well. A GnRH II receptor mRNA is expressed in human sperm (34), although the functional protein is yet to be defined. We have localized GnRH II to the testis, epididymis, and seminal vesicle and affected sperm function (Siler-Khodr et al., in preparation). These findings suggest that GnRH II may play a role in sperm development and function. In addition, we have also observed the production of GnRH II by the baboon fallopian tubes (Siler-Khodr et al., in preparation). These findings suggest a role for GnRH activity on tubal function.

Another site of GnRH II action may be the uterus, having a direct effect on the priming of the endometrium for implantation. Kang et al. (26) have demonstrated the expression of the mRNA for GnRH II in the human endometrium and its higher expression during the luteal phase of the cycle. We have observed high-affinity binding sites for this GnRH II analog, as well as an action of this analog on the stromal and epithelial endometrial production of prostaglandin E and prolactin from primary cell cultures (Siler-Khodr, in preparation). An interaction of this GnRH activity at endometrial and chorionic tissues or directly on differentiating trophoblasts may also be involved.

In our recent studies, we have demonstrated that the human placenta produces GnRH II and expresses high-affinity GnRH II binding sites (24). GnRH II and this GnRH II analog regulate hCG, progesterone, and prostaglandin E production from human placental explants (24). However, in the present study using this low dose of GnRH II analog, we found no abortifacient action in the three early pregnancies exposed to GnRH II analog when administered during the mid- to late luteal phase of the cycle. Each of these pregnancies resulted in normal liveborns.

A low-affinity receptor for GnRH I (µM) has been reported in the ovary, endometrium, and placenta, but this low-affinity binding seems much too low to account for the activity we have observed in these studies using 200 pM of this GnRH II analog. We have recently reported a second binding site in the baboon ovary and in human chorionic tissues with much higher affinity for GnRH II and its analogs. Millar et al. (35) have identified a GnRH II receptor in a variety of human tissues. Although these investigators initially speculated that the receptor’s function should be vestigial, they have more recently reported the expression of mRNA for the GnRH II receptor in the marmoset and human tissues (36). van Biljon et al. (34) have localized the GnRH II receptor transcript to human sperm. The expression of the GnRH II mRNA in human endometrial and ovarian cell lines, which are responsive to GnRH II, have also been reported (37). However, the nature of the functional GnRH II receptor remains to be established, because the mRNA in the human contains a stop codon.

The GnRH II analog used in these studies was designed to be resistant to circulating, ovarian, and chorionic enzymatic activities. We have previously defined a chorionic postproline peptidase, which rapidly degrades GnRH I and GnRH II (at one seventh the rate of GnRH I) (24, 28), and a similar activity we have observed in baboon ovarian and uterine extracts (10). Thus, the GnRH II analog used in these studies was designed to be stable in the presence of these activities as well as circulating endopeptidases. This analog’s stability, together with its high affinity for the GnRH II receptor in the reproductive tract and chorionic tissues (100-fold that of GnRH I), should effect a potent, sustained GnRH II activity.

These studies demonstrate the contraceptive activity of this GnRH II analog when administered in the fertile periovulatory-early luteal phase of the cycle. The presence of high-affinity, specific binding sites and activity of GnRH II or its analog at nM concentrations in reproductive tissues supports the possibility that many sites of action for GnRH II and its analogs, including the ovary, the ovum, the male reproductive tract, the sperm, the fallopian tube, the endometrium, and/or the trophoblast, may be involved in GnRH II regulation of reproductive physiology.


   Acknowledgments
 
The skilled technical assistance of Ms. Susan Coulthart and Ms. Shari Matyszczyk is gratefully acknowledged. We acknowledge the support of Southwest Genetics Professional Association for allowing us the use of their facilities. We also acknowledge Dr. A. Parlow and the National Pituitary Agency for making available the reagents for the LH assay and Dr. Gary Hodgens for his gift of reagents for the mCG RIA.


   Footnotes
 
This work was supported by the Consortium for Industrial Collaboration in Contraceptive Research (CICCR) Program of the Contraceptive Research and Development Program Foundation.

Views expressed do not necessarily reflect those of CONRAD or CICCR.

Abbreviations: hCG, Human chorionic gonadotropin; mCG, monkey chorionic gonadotropin.

Received January 13, 2004.

Accepted May 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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K. Morgan, R. Sellar, A. J. Pawson, Z.-L. Lu, and R. P. Millar
Bovine and Ovine Gonadotropin-Releasing Hormone (GnRH)-II Ligand Precursors and Type II GnRH Receptor Genes Are Functionally Inactivated
Endocrinology, November 1, 2006; 147(11): 5041 - 5051.
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