The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses

Rebecka Amodei; Sonnet S Jonker; William Whitler; Charles T Estill; Charles E Roselli

doi:10.1210/endocr/bqab262

. 2021 Dec 27;163(2):bqab262. doi: 10.1210/endocr/bqab262

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses

Rebecka Amodei ¹, Sonnet S Jonker ², William Whitler ⁴, Charles T Estill ^3,⁴, Charles E Roselli ^1,^✉

PMCID: PMC8760895 PMID: 34958103

Abstract

The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHβ-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Keywords: GnRH, testosterone, hypothalamus, anterior pituitary, fetus, gonadotropin

Male-typical differentiation of the brain and behavior depends on adequate production of testosterone (T) by the fetal testis within a specific “masculinization” programming window of gestation (1). This is referred to as the critical period for sexual differentiation of the brain. In short gestation altricial mammals such as rodents, this occurs during a perinatal window that begins in late gestation and extends into the first week of life. In long gestation precocial species such as primates and sheep, the critical period lasts for several weeks during the early part of the second trimester of gestation. In males of all species, the beginning of the critical period for brain differentiation occurs after the testicular Leydig cells develop and begin to secrete T (2). It also takes place after and is separate from the time when the genitalia have committed to masculinization (3,4). During this period T levels in blood rise to levels that are 2- to 10-fold higher in males than in females (5-8). However, the exact time when the hypothalamic-pituitary-gonadal axis is established and the pituitary gonadotrophin and testicular T secretion becomes dependent on control by hypothalamic gonadotropin-releasing hormone (GnRH) is not fully elucidated. In humans, placental human chorionic gonadotropin (hCG) initially plays a larger role than luteinizing hormone (LH) in stimulating T production by the Leydig cells. However, T secretion comes under fetal hypothalamic-pituitary control between 12 and 15 weeks of gestation, when LH secretion takes over for placental hCG (9). This transition is evident in GnRH/LH-deficient anencephalic male fetuses, in whom normal masculinization of the reproductive tract occurs while high levels of hCG are present, but further growth of the penis is hindered by the lack of LH and T later in gestation when hCG levels decrease (10). Similarly, the clinical condition of hypogonadotrophic hypogonadism resulting from one of several genetic mutations that cause deficiencies in GnRH migration or signaling is characterized by micropenis at birth indicating that T secretion was inadequate due to gonadotropin deficiency (11). Treatment of fetal cynomolgus monkeys with a long-acting GnRH agonist reduces penile length and testis size at birth suggesting that gonadotropins are also required for T secretion in fetal nonhuman primates (12). Two distinct perinatal surges of T have been identified in rodents. The first takes place late in gestation and is followed by a second surge occurring within 2 hours after birth. The prenatal rise in T occurs independently of GnRH/LH signaling (13,14). In contrast, data support both a GnRH-dependent (14-16) and independent mechanism for the immediate postnatal rise of T (17-19). There is also evidence that kisspeptin neurons project to and regulate the activity of GnRH neurons in the neonate and that sexual differentiation of the rodent brain requires kisspeptin-GnRH signaling (20-22). One explanation that may resolve the role GnRH plays in the postnatal T surge proposes a bimodal mechanism whereby initially GnRH-independent T secretion activates preoptic kisspeptin neurons to then stimulate the GnRH-dependent phase of T secretion (23).

In sheep, the anatomical, neural, and hormonal components of a functional hypothalamic-pituitary-gonadal axis are in place by gestational day (GD) 60; term pregnancy = ~147 ± 3 days (24). This is the time of onset for the critical period during which T masculinizes the ovine sexually dimorphic nucleus of the preoptic area (4). Whether GnRH neuron activity regulates LH and T secretion during this time remains relatively unexplored. Our previous study demonstrated that kisspeptin acts through GnRH neurons to stimulate a rise in LH secretion that is accompanied by an increase in T secretion (25). We reason that removing outputs from GnRH neurons could provide definitive evidence that GnRH signaling drives testicular steroidogenesis during this early period of gestation. So far, this kind of functional test has only been performed using GnRH agonist treatments in older sheep fetuses (26,27). Thus, the goal of the current research was to examine the role that GnRH signaling plays in the regulation of the pituitary-gonadal axis during the critical period of brain sexual differentiation in sheep. For this reason, we employed the long-acting GnRH antagonist degarelix to effectively curtail GnRH neuron activity by causing desensitization of GnRH receptors in pituitary gonadotrophs and determine whether this manipulation suppresses LH and T secretion in male lamb fetuses.

Materials and Methods

Animals

Time-bred mixed Western breeds of ewes (Ovis aries) and their fetuses were used in this study. Term pregnancy in these breeds is ~147 days. The sheep were purchased from the Oregon State University Sheep Center (Corvallis, OR, USA) or Agna (Salem, OR, USA). During our study, the ewes were maintained indoors with a fixed lighting cycle (lights on 6:00 am to 8 pm). All experimental protocols were conducted in accordance with National Institutes of Health (NIH) policy on the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Oregon Health and Science University and Oregon State University.

Fetal Injections

Twenty-four-hour fasted ewes carrying GD 62 fetuses were anesthetized with a jugular intravenous injection of diazepam (0.12 mg/kg; Abbott Laboratories, Abbott Park, IL, USA) and ketamine (4.4 mg/kg; Zoetis, Parsippany, NJ, USA). An endotracheal tube was placed and the ewe was maintained under anesthesia by ventilation with isoflurane (1.5-2.5%) in oxygen. Maternal heart rate, temperature, arterial oxygen saturation, respiratory rate, end tidal CO2, and adequacy of ventilation were continuously monitored during anesthesia. The ewe’s abdomen was opened through either a mid-line incision or left flank approach using a sterile surgical technique. The uterus was exposed and opened in layers, maintaining the fetal membranes in close approximation to the endometrium. The lamb fetus was exteriorized, sexed and given bilateral subcutaneous injections along the spine of degarelix acetate [Firmagon®, Ferring Pharmaceuticals, Parsippany, NJ, USA; 2 mg/500 µL (~35 mg/kg)]. Control animals were given injections of 5% mannitol vehicle (500 µL). The tail was docked to later confirm treatment. The lamb was returned into the amnion sac along with 1 million U Penicillin G (Pfizer, New York, NY, USA) and 2 mg of ciprofloxacin (Fisher Scientific, Pittsburgh, PA, USA). The uterus was sutured closed with an Utrecht suture pattern. When twins were carried by the ewe, the procedure was repeated for the second fetus. The uterus was returned into the abdomen and the individual muscle layers of abdomen or flank closed with continuous absorbable suture. The ewe’s skin was stapled and sprayed with Alushield (Neogen, Lexington, KY, USA) and a transdermal 100 µg/hour fentanyl patch (Duragesic, Janssen Labs) applied.

Fetal Catheterization

Two to 3 weeks after the first surgery (ie, GD 76 or 85), maternal ewes were anesthetized and underwent a second sterile surgery as previously described to place arterial and venous catheters into their treated fetuses. The uterus was opened as before, the fetal head and neck were exteriorized, and medical grade micro-vinyl catheters (0.58 mm internal diameter × 0.99 mm external diameter; Scientific Commodities Inc, Lake Havasu City, AZ, USA) filled with heparinized saline were placed into a fetal carotid artery and jugular vein and secured by suturing to the fetal skin. The fetus was returned into the amnion within the uterus, and the incisions were closed. The catheters were fed through the uterine and abdominal incisions to provide access for drug administration and blood sampling. When twins were carried by the ewe, the procedure was repeated for the second fetus.

GnRH Challenge

GnRH (Cystorelin; Boehringer Ingelheim Animal Health USA Inc., Duluth, GA, USA) was administered as a sterile bolus injection (25 µg/500 µL) into the jugular vein catheter. Fetal blood samples (0.9 mL) were collected from the carotid artery catheter 15 minutes before and just prior to drug administration (0 minutes) and then at 30-minute intervals for the next 2 hours. The entire experiment was performed while the sheep were anesthetized. Arterial blood gases and pH were monitored to assure that the fetus remained in good health throughout the experiment. All experiments were performed between 9:00 am and 12:.00 pm.

Hormone Assays

LH concentrations were measured in plasma by radioimmunoassay using reagents provided by the National Hormone and Peptide Program (Torrance, CA, USA) as previously described (28). LH assay sensitivity averaged 0.020 ng/mL (NIH S19) with intra- and interassay coefficients of variations equal to 4.1% and 7.4%, respectively. Total T levels were measured by the Oregon Health and Science University Endocrine Technologies Core using ultra-high-performance liquid chromatography-heated electrospray ionization-tandem triple quadrupole mass spectrometry on a Shimadzu Nexera-LCMS-8050 platform (Kyoto, Japan). T assay sensitivity averaged 10 pg/mL and the intra- and interassay variations were 8.1% and 10%, respectively.

Tissue Collection

Fetuses were surgically delivered immediately after blood sampling was complete and while the pregnant ewes remained anesthetized. A final blood sample was taken from the umbilical artery and then the umbilical cord was tied and cut. Fetuses were weighed and measured and their sex recorded. Brains, pituitaries, and gonads were removed, divided, and frozen on dry ice for subsequent RNA extraction or immersion fixed overnight at 4°C in 4% paraformaldehyde, cryoprotected in 20% sucrose, and frozen for subsequent immunohistochemistry.

Quantitative Reverse Transcription Real-time Polymerase Chain Reaction

Total RNA was extracted from anterior pituitaries and gonads using an RNeasy Lipid Tissue Mini Kit (Qiagen) following the manufacturer’s instructions. The RNA was quality tested and converted to complimentary DNA using the SuperScript™ III First-Strand Synthesis System (Invitrogen, Waltham, MA, USA). Real-time polymerase chain reactions were run in triplicate using PowerSYBR Green Master Mix (Invitrogen) and previously validated sheep-specific primer sets (11). All reactions were run in a Quant Studio 7 Flex Thermal Cycler (Applied Biosystems, Life Technologies, Eugene, OR, USA). Quantification of gene expression was performed by the delta delta Ct method, using complimentary DNA from pooled GD 85 to 100 anterior pituitary and gonadal tissues as calibrators and normalized against the reference gene GAPDH. Data are reported as the fold difference relative to the mean for male controls.

Immunohistochemistry

Fixed anterior pituitaries were sectioned on a cryostat at 20 µm, thaw mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA, USA) and stored at −20°C until processing. Fluorescent immunohistochemistry (IHC) was performed on 4 consecutive sections from each animal using a protocol described previously (29). Briefly, sections were washed in 0.1 M phosphate buffer (PB, pH 7.4) for 20 minutes and incubated at 4°C overnight with guinea pig anti-c-fos antibody (1:500; Synaptic Systems, cat no. 226 004, Gottingen, Germany) (30) and rabbit anti-Ovine LHβ antibody (1:1000; A. F. Parlow National Hormone and Peptide Program cat no. AFP—oLHB) (31). The following day, sections were washed in PB and then incubated for 90 minutes at room temperature in goat anti-rabbit fluorescein isothiocyanate, 1:250 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and donkey anti-guinea pig-Cy3, 1:250 (Jackson ImmunoResearch Laboratories). After rinsing in PB, the slides were coverslipped with Prolong Gold Anti-Fade Reagent with 4′,6-diamidino-2-phenylindole (DAPI).

Microscopy and Image Analysis

For IHC, imaging was performed with epifluorescence microscopy using a Zeiss Axioplan 2 microscope (Carl Zeiss, Inc, Germany). Low power (20×) images of the pituitary were taken at a constant exposure from IHC and DAPI-stained sections. Composite 8-bit digital images were then imported into Fiji software (NIH Image J) (32), and after merging channels, colors were assigned and the color balance was adjusted for optimal presentation. Slides were coded during microscopy, and all analysis was performed under blinded conditions to avoid experimenter bias. For single and double antigen staining, cell bodies were identified by fluorescent cytoplasmic staining that also exhibited nuclear DAPI staining. The total number of single- and double-labeled (LHβ and c-fos) cells was counted manually using the multipoint tool in Fiji. The micrographs presented in this article were taken using the ApoTome system and Zen Pro Axiovision Imaging software (Carl Zeiss, Inc).

Statistics

Data were analyzed using GraphPad Prism version 9.2 (GraphPad Software, San Diego, CA, USA). Response parameters to the GnRH challenge were calculated using area under the curve (AUC) analysis with the baseline calculated as the average of the −30 and 0 hours. values. All data are presented as mean ± SE of the mean, with P < 0.05 considered statistically significant. Statistical evaluations of LH and T response curves were carried out with 1-way repeated measures analysis of variance (ANOVA) followed by a Dunnett’s post hoc comparison tests. Sex × Treatment comparisons were evaluated with 2-way ANOVA followed by Tukey’s multiple comparison post hoc tests. Data were log₁₀ transformed when necessary to equalize variances among groups. Unpaired Student’s t tests were used to compare data when only 2 groups, control vs degarelix-treated, were analyzed.

Results

Effect of Degarelix Treatment on Baseline and GnRH-stimulated LH and T Release

Intravenous administration of 25 µg of GnRH elicited a rapid LH response in midgestation male and female control lamb fetuses (Fig. 1A) and provoked a T response in both control and degarelix-treated males (Fig. 1B). Treatment with degarelix suppressed LH secretion in males and females and T responses in males. T concentrations in females were measured as a control to judge the completeness of T suppression in males. Baseline concentrations of LH were significantly higher (P < 0.05) in control females than in males and significantly (P < 0.05) reduced in both sexes by degarelix treatment (Fig. 2A). Baseline concentrations of T were significantly (P < 0.05) greater in males than in females and significantly (P < 0.05) reduced to levels as low as females in degarelix-treated males (Fig. 3A). The overall LH secretory response elicited by GnRH, estimated as the AUC, was not different between control males and females and was significantly (P < 0.05) lower in degarelix-treated fetuses of both sexes (Fig. 2B). The AUC for the T response was significantly (P < 0.05) greater in control males than in females and significantly (P < 0.05) reduced in degarelix-treated males but not females (Fig. 3B). The peak LH response did not differ between the sexes and was significantly (P < 0.05) suppressed in both sexes after degarelix treatment (Fig. 2C). The peak T response was significantly (P < 0.05) greater in males than in females and significantly (P < 0.05) reduced in degarelix-treated males but unaltered in females (Fig. 3C). It is interesting to note that despite a significant decrease in a baseline and peak LH and T concentrations, degarelix-treated fetuses were still capable of responding with a comparable fold increase to exogenous GnRH.

Figure 1. — Changes in the concentration of luteinizing hormone (A) and testosterone (B) in control (CTL) and degarelix (DG)-treated male and female lamb fetuses studied at 76 to 85 days after intravenous bolus injection of 25-µg gonadotropin-releasing hormone. Data (mean ± SE of the mean) were analyzed by 1-way repeated measures analysis of variance followed by Dunnett’s multiple comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001 vs baseline values.

Figure 2. — Baseline luteinizing hormone (LH) (A) and LH response measures (B and C) to bolus intravenous injection of gonadotropin-releasing hormone in control (CTL) and degarelix (DG)-treated male and female lamb fetuses. Data were analyzed by 2-way analysis of variance followed by Tukey’s multiple comparison tests. Bars (mean ± SE of the mean) with different superscripts differ significantly (P < 0.05). Abbreviation: AUC, area under the curve.

Figure 3. — Baseline testosterone (A) and testosterone response measures (B and C) to bolus intravenous injection of gonadotropin-releasing hormone in control (CTL) and degarelix (DG)-treated male and female lamb fetuses. Data were analyzed by 2-way analysis of variance followed by Tukey’s multiple comparison tests. Bars (mean ± SE of the mean) with different superscripts differ significantly (P < 0.05). Abbreviation: AUC, area under the curve.

Effect of Degarelix Treatment on Pituitary Gonadotropin Subunit Expression and c-fos Response to GnRH

Figure 4 illustrates the effects of degarelix treatment on the expression of gonadotropin subunit and GnRH receptor messenger RNA (mRNA) in the fetal anterior pituitary. Levels of pituitary LHβ subunit RNA were significantly (P < 0.05) greater in control females than in males. Treatment with degarelix significantly (P < 0.05) reduced pituitary expression of the LHβ mRNA by 95% in both sexes when compared to controls (Fig. 4A). Levels of follicle-stimulating hormone (FSH) β and the common α-subunit (CαS) mRNA were significantly (P < 0.05) higher in females than in males. Degarelix treatment significantly (P < 0.05) suppressed FSHβ and CαS mRNA levels in females but not in males, which were already low (Fig. 4B and 4C). GnRH receptor expression was not different between sexes nor altered by degarelix treatment (Fig. 4D). To confirm that degarelix treatment reduced gonadotroph sensitivity to GnRH, we compared the extent to which LHβ cells colocalized c-fos after GnRH stimulation (Fig. 5C). The percentage colocalization was significantly (P < 0.01) greater in control fetuses (ie, 76 ± 3.0%) than in degarelix-treated fetuses (ie, 35 ± 1%). There was no difference in the number of LHβ-immunoreactive cells counted per section between control and degarelix-treated fetuses (Fig. 5D).

Figure 5. — Photomicrographs of pituitary sections stained for luteinizing hormone (LH) β (green) and c-fos (red) from control (CTL) (A) and degarelix (DG)-treated (B) lamb fetuses. White arrow indicate examples of c-fos-positive LH cells. Open triangles indicate examples of c-fos-negative LH cells. White scale bar = 20 µm. Percentage [mean ± SE of the mean (SEM)] of LHβ-positive cells containing c-fos-positive nuclei (C). Total number (mean ± SEM) of LHβ-positive cells in 4 consecutive 20-µm sections from pituitary (D). Data were analyzed by unpaired Student t tests. *P<0.05; **P<0.01.

Effect of Degarelix Treatment on the Expression of Steroidogenic Enzymes and LH receptor in the Gonads

Figure 6 illustrates that degarelix treatment downregulates expression of key enzyme mRNAs needed for androgen synthesis by the fetal gonads. There was a significant (P < 0.05) decrease in the expression of steroid acute regulatory protein (StAR) and Cyp 17 (Fig. 6A and 6B). The expression of StAR and cytochrome P450 (Cyp) 17 in the fetal ovary was ~1% of that in the testis and unaffected by degarelix treatment. Neither expression of testicular Cyp 11 nor ovarian Cyp 19 (ie, aromatase) mRNA expression was different between treatment groups in the testes and ovaries, respectively (Fig. 6C and 6D). LH receptor (expression was not altered in either the testis or ovary, but was 10-fold lower in ovary (Fig. 6E).

Effect of Degarelix Treatment on Physical Measurements

As shown in Table 1, degarelix treatment had no effect on body weight, brain weight, or crown-rump length. There was, as expected, a significant (P < 0.05) sex difference in the ratio of ano-genital distance to ano-umbilical distance but no effect of degarelix treatment on this measure.

Table 1.

Effects of degarelix injections at GD62 on physical measurements in GD76-85 ovine fetuses

Measure	Treatment
	CTL male	DG male	CTL female	DG female
Body wt, g	566 ± 42	401 ± 55	518 ± 18	412 ± 80
Brain wt, g	12 ± 0.5	11 ± 1.7	13 ± 0.7	11 ± 1.9
CRL, cm	27 ± 0.4	25 ± 1.7	27 ± 0.4	24 ± 2.5
AGD/AUD	0.9 ± 0.03	0.8 ± 0.02	0.1 ± 0.005	0.1 ± 0.01
n	3	4	5	2

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Data are means ± SE of the mean.

Abbreviations: AGD/AUD, ratio of anogenital distance to anoumbilical distance; CTL, control; CRL, crown-rump length; DG, degarelix; wt, weight.

Discussion

We have shown that treatment of lamb fetuses early in the second trimester with the long-acting GnRH antagonist degarelix acetate produced profound suppression of the gonadotrophic axis. This was characterized by reduced baseline and GnRH-stimulated LH secretion, decreased gonadotrophin subunit mRNA expression, and a suppressed gonadotroph c-fos response to GnRH challenge. These changes were evident 2 to 3 weeks after administering subcutaneous degarelix injections to fetuses. The resultant pituitary desensitization and consequent inhibition of gonadotrophic support was accompanied by significantly suppressed basal T concentrations in degarelix-treated males, such that basal concentrations in males were not different from concentration in control females. Degarelix treatment also significantly reduced GnRH-stimulated T secretion in males and downregulated expression of genes in the androgen synthesis pathway. These results demonstrate that GnRH antagonist treatment is an effective strategy for interrupting androgen exposure in mid-gestation male lamb fetuses.

Baseline levels of LH and steady state levels of pituitary LHβ mRNA expression were higher in females than in males, yet there were no differences between the sexes in the parameters of GnRH-stimulated LH. All measures were suppressed by degarelix treatment, indicating that GnRH input regulates LH synthesis and secretion in male and female fetuses at this stage in gestation. The observation that immunohistochemical colocalization of c-fos with LHβ was reduced indicated that antagonist treatment decreased the sensitivity of LHβ-containing gonadotrophs to GnRH. The expression of FSHβ and the CαS was greater in females than in males and suppressed in females after degarelix treatment. These results suggest that GnRH regulates both LH and FSH synthesis and secretion in ovine fetuses early in gestation. The observed sex difference in steady state levels of gonadotropin mRNA agrees with our previous findings (33). The greater sex difference in FSHβ vs LHβ mRNA expression suggest that the testes suppress FSH synthesis in large part by acting on the pituitary, possibly through inhibin secretion, whereas negative feedback on LH appears to be exerted predominantly through T feedback at the level of the hypothalamus. At this age, inhibin is expressed by fetal testes but not ovaries (34,35), and FSH is elevated by gonadectomy in males but not in females (36). Although we did not measure plasma FSH in the present study, previous studies reported that plasma LH and FSH between gestational ages 71 to 110 days are higher in females than in males (37,38).

Our current study found that plasma concentrations of T were 12-fold greater in male lamb fetuses than in females and the expression of mRNA coding for androgen biosynthesis enzymes were 10- to 100-fold higher in the fetal testis than in ovaries. This agrees with reports that the fetal ovary has very little steroidogenic capacity in early gestation (8,39). Administration of degarelix decreased basal and GnRH-stimulated T concentrations in males but not females in which it remained at baseline levels. Antagonist treatment also suppressed the expression of mRNA for StAR protein, which facilitates transfer of cholesterol across the mitochondrial membrane and is the rate-limiting step in steroidogenesis, and for Cyp 17 an essential hydroxylase in the pathway for T synthesis.

We observed no effect of degarelix treatment on steroidogenic enzyme mRNA expression in ovary, including Cyp 19 aromatase, a key enzyme for estrogen biosynthesis. The T response to a bolus injection of GnRH and its suppression after degarelix treatment coincides with parallel changes in plasma LH supporting the existence of a functioning hypothalamic-pituitary-testicular axis at this gestational age. By contrast, the low steroidogenic activity in ovaries and the lack of response to degarelix suggest that although the gonadotropic axis functions in early gestation, it does not play a central role in ovarian function or maturation at this stage of gestation. This is not very surprising since it was reported that suppression of the fetal gonadotropic axis from day 70 of gestation until birth had no effect on ovarian development but severely impairs testicular growth and steroidogenesis (40). Ex vivo studies support the hypothesis that during sheep gestation steroid hormones play autocrine/paracrine roles in ovarian follicular and vascular development (39). The ovary was not the central focus of the current study, but it may be possible in future studies using degarelix to address more directed questions regarding the role of gonadotropins in the maturation of the fetal ovary.

The reduction of steroidogenic enzyme expression in testis most likely resulted from inhibition of gonadotrophic support to Leydig cells. Previous studies found that LH increases T synthesis in short-term fetal testis explant cultures and demonstrated increased exposure to androgens prenatally suppresses LH in conjunction with downregulation of the genes involved with T synthesis (41,42). However, we cannot rule out the possibility that degarelix exerts direct effects on the fetal testis. Evidence in mice suggests that degarelix inhibits the ability of Leydig cells to produce T by directly suppressing the expression of genes involved in steroidogenesis such as StAR, Cyp 11, and 17β hydroxysteroid dehydrogenase (43). Thus, further research is needed to more fully understand the control of steroidogenesis in the fetal lamb testis and determine whether it can be directly suppressed by degarelix.

The effects of degarelix acetate on the male lamb fetus are in agreement with previous studies in adult rats, goats, and primates (44-46). Upon subcutaneous administration, degarelix forms a gel that results in a sustained release of the compound into the circulation, immediately blocking GnRH receptors in the pituitary and inducing a rapid dose-dependent suppression of the pituitary-gonadal axis as revealed by the decrease in plasma LH and T levels lasting 4 to 8 weeks. Studies in goats demonstrated that the hormonal effects of degarelix-acetate are completely reversed after treatment ends (45). Repeated monthly treatment of goats is effective as a method of chemical castration reducing sperm count as well as hormone secretion (47). The rapid and sustained ability of degarelix to suppress circulating T concentrations and its low toxicity has led to Food and Drug Administration–approved use for clinical treatment of hormone-responsive prostate cancer (48).

In summary, our results suggest that treatment with degarelix is an effective way to inhibit fetal gonadotropin secretion and could be useful to investigate hypothalamic-pituitary-testicular function during sexual differentiation of the brain. The use of degarelix has distinct advantages over previous approaches used to block the fetal gonadotropic axis (26,40,49). These include the rapidity of its effects. In adult goats, LH pulse frequency is decreased significantly within 6 hours after administration, and T is already reduced within an hour after treatment (45). By contrast, GnRH agonists produce an initial flare of LH and T secretion and both agonist treatment and passive immunization take several days before hormone levels fall (26,49). To suppress the gonadotropic axis for extended time periods, agonists must be repeatedly injected or administered as implants, whereas in the current study, a single injection of degarelix resulted in suppression of basal and GnRH-stimulated LH and T levels for 2 to 3 weeks and possibly longer (unpublished observation). This same dose of degarelix given to adult male goats reduced blood T levels for 2 months (45).

These results demonstrate that pharmacologic inhibition of LH secretion early in gestation results in significant contemporaneous deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T secretion during a critical prenatal period of early development when the sheep brain is masculinized. We cannot say this has postnatal consequences, but it is biologically plausible that it could. Thus, longer term studies, which are currently underway, are warranted to assess the effect prenatal treatment with GnRH antagonists has on adult sexual behaviors and fertility.

Acknowledgments

We thank the many Oregon State University students who cared for the sheep used in this study. We thank Mary Smallman of Oregon State University and Lucy Cross of Agna for supplying the healthy sheep used in this study. We thank Sarah M. M. Alaniz and Dr. Samantha Louey for assistance with surgeries at Oregon Health & Science University. Hormone measurements were performed by Dr. Robert Goodman at West Virginia University and the Endocrine Technology and Support Core at the Oregon National Primate Research Center.

Financial Support

This work was supported by National Institutes of Health grants R01OD011047 to C.E.R.; R01HL142483 to S.J; and P51 OD011092 to the Oregon National Primate Research Center.

Disclosure Statement

The authors have nothing to disclose.

Data Availability

Some or all data sets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

References

1. MacLusky NJ, Naftolin F. Sexual differentiation of the central nervous system. Science. 1981;211(4488):1294-1302. [DOI] [PubMed] [Google Scholar]
2. O’Shaughnessy P. Testicular development. In: Plant TM, Zeleznik AJ, eds. Knobil and Neill’s Physiology of Reproduction. 4th ed. Academic Press; 2015:567-594. [Google Scholar]
3. Goy RW, Bercovitch FB, McBrair MC. Behavioral masculinization is independent of genital masculinization in prenatally androgenized female rhesus macaques. Horm Behav. 1988;22(4):552-571. [DOI] [PubMed] [Google Scholar]
4. Roselli CE, Estill CT, Stadelman HL, Meaker M, Stormshak F. Separate critical periods exist for testosterone-induced differentiation of the brain and genitals in sheep. Endocrinology. 2011;152(6):2409-2415. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Resko JA, Roselli CE. Prenatal hormones organize sex differences of the neuroendocrine reproductive system: observations on guinea pigs and nonhuman primates. Cell Mol Neurobiol. 1997;17(6):627-648. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Baum MJ, Woutersen PJ, Slob AK. Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females. Biol Reprod. 1991;44(5):747-751. [DOI] [PubMed] [Google Scholar]
7. Scott HM, Mason JI, Sharpe RM. Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. Endocr Rev. 2009;30(7):883-925. [DOI] [PubMed] [Google Scholar]
8. Pomerantz DK, Nalbandov AV. Androgen level in the sheep fetus during gestation. Proc Soc Exp Biol Med. 1975;149(2):413-416. [DOI] [PubMed] [Google Scholar]
9. Choi J, Smitz J. Luteinizing hormone and human chorionic gonadotropin: distinguishing unique physiologic roles. Gynecol Endocrinol. 2014;30(3):174-181. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Bearn JG. Anencephaly and the development of the male genital tract. Acta Paediatr Acad Sci Hung. 1968;9(2):159-180. [PubMed] [Google Scholar]
11. Bouvattier C, Maione L, Bouligand J, Dodé C, Guiochon-Mantel A, Young J. Neonatal gonadotropin therapy in male congenital hypogonadotropic hypogonadism. Nat Rev Endocrinol. 2012;8(3):172-182. [DOI] [PubMed] [Google Scholar]
12. Liu L, Cristiano AM, Southers JL, et al. Effects of pituitary-testicular axis suppression in utero and during the early neonatal period with a long-acting luteinizing hormone-releasing hormone analog on genital development, somatic growth, and bone density in male cynomolgus monkeys in the first 6 months of life. J Clin Endocrinol Metab. 1991;73(5):1038-1043. [DOI] [PubMed] [Google Scholar]
13. Nakamura S, Uenoyama Y, Ikegami K, et al. Neonatal kisspeptin is steroid-independently required for defeminisation and peripubertal kisspeptin-induced testosterone is required for masculinisation of the brain: a behavioural study using kiss1 knockout rats. J Neuroendocrinol. 2016;28(10). Doi: 10.1111/jne.12409 [DOI] [PubMed] [Google Scholar]
14. O’Shaughnessy PJ, Baker P, Sohnius U, Haavisto AM, Charlton HM, Huhtaniemi I. Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology. 1998;139(3):1141-1146. [DOI] [PubMed] [Google Scholar]
15. Corbier P, Kerdelhue B, Picon R, Roffi J. Changes in testicular weight and serum gonadotropin and testosterone levels before, during, and after birth in the perinatal rat. Endocrinology. 1978;103(6):1985-1991. [DOI] [PubMed] [Google Scholar]
16. Clarkson J, Busby ER, Kirilov M, Schütz G, Sherwood NM, Herbison AE. Sexual differentiation of the brain requires perinatal kisspeptin-GnRH neuron signaling. J Neurosci. 2014;34(46):15297-15305. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Slob AK, Ooms MP, Vreeburg JT. Prenatal and early postnatal sex differences in plasma and gonadal testosterone and plasma luteinizing hormone in female and male rats. J Endocrinol. 1980;87(1):81-87. [DOI] [PubMed] [Google Scholar]
18. McGivern RF, Hermans RHM, Handa RJ, Longo LD. Plasma testosterone surge and luteinizing hormone beta (LH-b) following parturition: lack of association in the male rat. Eur J Endocrinol. 1995;133(3):366-374. [DOI] [PubMed] [Google Scholar]
19. Poling MC, Kauffman AS. Sexually dimorphic testosterone secretion in prenatal and neonatal mice is independent of kisspeptin-Kiss1r and GnRH signaling. Endocrinology. 2012;153(2):782-793. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Kumar D, Freese M, Drexler D, Hermans-Borgmeyer I, Marquardt A, Boehm U. Murine arcuate nucleus kisspeptin neurons communicate with GnRH neurons in utero. J Neurosci. 2014;34(10):3756-3766. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Kumar D, Periasamy V, Freese M, Voigt A, Boehm U. In utero development of kisspeptin/GnRH neural circuitry in male mice. Endocrinology. 2015;156(9):3084-3090. [DOI] [PubMed] [Google Scholar]
22. Kauffman AS, Park JH, McPhie-Lalmansingh AA, et al. The kisspeptin receptor GPR54 is required for sexual differentiation of the brain and behavior. J Neurosci. 2007;27(33):8826-8835. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Nakamura S, Watanabe Y, Goto T, Ikegami K, Inoue N, Uenoyama Y, Tsukamura H. Kisspeptin neurons as a key player bridging the endocrine system and sexual behavior in mammals. Front Neuroendocrinol. Published online October 29, 2021. Doi: 10.1016/j.yfrne.2021.100952 [DOI] [PubMed] [Google Scholar]
24. Brooks AN, Hagan DM, Sheng C, McNeilly AS, Sweeney T. Prenatal gonadotrophins in the sheep. Anim Reprod Sci. 1996;42(1-4):471-481. [Google Scholar]
25. Amodei R, Gribbin K, He W, et al. Role for kisspeptin and neurokinin B in regulation of luteinizing hormone and testosterone secretion in the fetal sheep. Endocrinology. 2020;161(4):1-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Clark SJ, Hauffa BP, Rodens KP, Styne DL, Kaplan SL, Grumbach MM. Hormone ontogeny in the ovine fetus: XIX: The effect of a potent luteinizing hormone-releasing factor agonist on gonadotropin and testosterone release in the fetus and neonate. Pediatr Res. 1989;25(4):347-352. [DOI] [PubMed] [Google Scholar]
27. Brooks AN, Currie IS, Gibson F, Thomas GB. Neuroendocrine regulation of sheep fetuses. J Reprod Fertil Suppl. 1992;45:69-84. [PubMed] [Google Scholar]
28. Niswender GD, Reichert LE Jr, Midgley AR Jr, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology. 1969;84(5):1166-1173. [DOI] [PubMed] [Google Scholar]
29. Dziennis S, Jia T, Rønnekleiv OK, Hurn PD, Alkayed NJ. Role of signal transducer and activator of transcription-3 in estradiol-mediated neuroprotection. J Neurosci. 2007;27(27):7268-7274. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.RRID:AB_2619946. https://antibodyregistry.org/search.php?q=AB_2619946
31.RRID:AB_2629449. https://antibodyregistry.org/search.php?q=AB_2629449
32. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-682. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Roselli CE, Reddy RC, Estill CT, et al. Prenatal influence of an androgen agonist and antagonist on the differentiation of the ovine sexually dimorphic nucleus in male and female lamb fetuses. Endocrinology. 2014;155(12):5000-5010. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Jarred RA, Cancilla B, Richards M, Groome NP, McNatty KP, Risbridger GP. Differential localization of inhibin subunit proteins in the ovine testis during fetal gonadal development. Endocrinology. 1999;140(2):979-986. [DOI] [PubMed] [Google Scholar]
35. Engelhardt H, Harkness LM, Thomas GB, Brooks AN, McNeilly AS, Baird DT. Expression of inhibin alpha- and beta A-subunit mRNA and protein in the fetal sheep ovary throughout gestation. Mol Cell Endocrinol. 1995;107(2):141-147. [DOI] [PubMed] [Google Scholar]
36. Matwijiw I, Faiman C. Control of gonadotropin secretion in the ovine fetus. IV. Male-specific entrainment of the hypothalamic control of luteinizing hormone secretion by testosterone in the female ovine fetus. Endocrinology. 1991;129(3):1447-1451. [DOI] [PubMed] [Google Scholar]
37. Sklar CA, Mueller PL, Gluckman PD, Kaplan SL, Rudolph AM, Grumbach MM. Hormone ontogeny in the ovine fetus. VII. Circulating luteinizing hormone and follicle-stimulating hormone in mid- and late gestation. Endocrinology. 1981;108(3):874-880. [DOI] [PubMed] [Google Scholar]
38. Foster DL, Roach JF, Karsch FJ, Norton HW, Cook B, Nalbandov AV. Regulation of luteinizing hormone in the fetal and neonatal lamb. I. LH concentrations in blood and pituitary. Endocrinology. 1972;90(1):102-111. [DOI] [PubMed] [Google Scholar]
39. Juengel JL, Sawyer HR, Smith PR, et al. Origins of follicular cells and ontogeny of steroidogenesis in ovine fetal ovaries. Mol Cell Endocrinol. 2002;191(1):1-10. [DOI] [PubMed] [Google Scholar]
40. Thomas GB, McNeilly AS, Gibson F, Brooks AN. Effects of pituitary-gonadal suppression with a gonadotrophin-releasing hormone agonist on fetal gonadotrophin secretion, fetal gonadal development and maternal steroid secretion in the sheep. J Endocrinol. 1994;141(2):317-324. [DOI] [PubMed] [Google Scholar]
41. Connolly F, Rae MT, Bittner L, Hogg K, McNeilly AS, Duncan WC. Excess androgens in utero alters fetal testis development. Endocrinology. 2013;154(5):1921-1933. [DOI] [PubMed] [Google Scholar]
42. Roselli CE, Amodei R, Gribbin KP, Corder K, Stormshak F, Estill CT. Excess testosterone exposure alters hypothalamic-pituitary-testicular axis dynamics and gene expression in sheep fetuses. Endocrinology. 2016;157(11):4234-4245. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Taniguchi H, Katano T, Nishida K, Kinoshita H, Matsuda T, Ito S. Elucidation of the mechanism of suppressed steroidogenesis during androgen deprivation therapy of prostate cancer patients using a mouse model. Andrology. 2016;4(5):964-971. [DOI] [PubMed] [Google Scholar]
44. Broqua P, Riviere PJ, Conn PM, Rivier JE, Aubert ML, Junien JL. Pharmacological profile of a new, potent, and long-acting gonadotropin-releasing hormone antagonist: degarelix. J Pharmacol Exp Ther. 2002;301(1):95-102. [DOI] [PubMed] [Google Scholar]
45. Hannan MA, Kawate N, Fukami Y, et al. Effects of long-acting GnRH antagonist, degarelix acetate, on plasma insulin-like peptide 3, testosterone and luteinizing hormone concentrations, and scrotal circumference in male goats. Theriogenology. 2017;88:228-235. [DOI] [PubMed] [Google Scholar]
46. Princivalle M, Broqua P, White R, et al. Rapid suppression of plasma testosterone levels and tumor growth in the dunning rat model treated with degarelix, a new gonadotropin-releasing hormone antagonist. J Pharmacol Exp Ther. 2007;320(3):1113-1118. [DOI] [PubMed] [Google Scholar]
47. Kawate N, Kanuki R, Hannan MA, Weerakoon WWPN. Inhibitory effects of long-term repeated treatments of a sustainable GnRH antagonist, degarelix acetate, on caprine testicular functions. J Reprod Dev. 2020;66(6):587-592. [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Kittai AS, Blank J, Graff JN. Gonadotropin-releasing hormone antagonists in prostate cancer. Oncology (Williston Park). 2018;32(12):599-602, 604. [PubMed] [Google Scholar]
49. Miller DW, Fraser HM, Brooks AN. Suppression of fetal gonadotrophin concentrations by maternal passive immunization to GnRH in sheep. J Reprod Fertil. 1998;113(1):69-73. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Some or all data sets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

[CIT0001] 1. MacLusky NJ, Naftolin F. Sexual differentiation of the central nervous system. Science. 1981;211(4488):1294-1302. [DOI] [PubMed] [Google Scholar]

[CIT0002] 2. O’Shaughnessy P. Testicular development. In: Plant TM, Zeleznik AJ, eds. Knobil and Neill’s Physiology of Reproduction. 4th ed. Academic Press; 2015:567-594. [Google Scholar]

[CIT0003] 3. Goy RW, Bercovitch FB, McBrair MC. Behavioral masculinization is independent of genital masculinization in prenatally androgenized female rhesus macaques. Horm Behav. 1988;22(4):552-571. [DOI] [PubMed] [Google Scholar]

[CIT0004] 4. Roselli CE, Estill CT, Stadelman HL, Meaker M, Stormshak F. Separate critical periods exist for testosterone-induced differentiation of the brain and genitals in sheep. Endocrinology. 2011;152(6):2409-2415. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0005] 5. Resko JA, Roselli CE. Prenatal hormones organize sex differences of the neuroendocrine reproductive system: observations on guinea pigs and nonhuman primates. Cell Mol Neurobiol. 1997;17(6):627-648. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0006] 6. Baum MJ, Woutersen PJ, Slob AK. Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females. Biol Reprod. 1991;44(5):747-751. [DOI] [PubMed] [Google Scholar]

[CIT0007] 7. Scott HM, Mason JI, Sharpe RM. Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. Endocr Rev. 2009;30(7):883-925. [DOI] [PubMed] [Google Scholar]

[CIT0008] 8. Pomerantz DK, Nalbandov AV. Androgen level in the sheep fetus during gestation. Proc Soc Exp Biol Med. 1975;149(2):413-416. [DOI] [PubMed] [Google Scholar]

[CIT0009] 9. Choi J, Smitz J. Luteinizing hormone and human chorionic gonadotropin: distinguishing unique physiologic roles. Gynecol Endocrinol. 2014;30(3):174-181. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0010] 10. Bearn JG. Anencephaly and the development of the male genital tract. Acta Paediatr Acad Sci Hung. 1968;9(2):159-180. [PubMed] [Google Scholar]

[CIT0011] 11. Bouvattier C, Maione L, Bouligand J, Dodé C, Guiochon-Mantel A, Young J. Neonatal gonadotropin therapy in male congenital hypogonadotropic hypogonadism. Nat Rev Endocrinol. 2012;8(3):172-182. [DOI] [PubMed] [Google Scholar]

[CIT0012] 12. Liu L, Cristiano AM, Southers JL, et al. Effects of pituitary-testicular axis suppression in utero and during the early neonatal period with a long-acting luteinizing hormone-releasing hormone analog on genital development, somatic growth, and bone density in male cynomolgus monkeys in the first 6 months of life. J Clin Endocrinol Metab. 1991;73(5):1038-1043. [DOI] [PubMed] [Google Scholar]

[CIT0013] 13. Nakamura S, Uenoyama Y, Ikegami K, et al. Neonatal kisspeptin is steroid-independently required for defeminisation and peripubertal kisspeptin-induced testosterone is required for masculinisation of the brain: a behavioural study using kiss1 knockout rats. J Neuroendocrinol. 2016;28(10). Doi: 10.1111/jne.12409 [DOI] [PubMed] [Google Scholar]

[CIT0014] 14. O’Shaughnessy PJ, Baker P, Sohnius U, Haavisto AM, Charlton HM, Huhtaniemi I. Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology. 1998;139(3):1141-1146. [DOI] [PubMed] [Google Scholar]

[CIT0015] 15. Corbier P, Kerdelhue B, Picon R, Roffi J. Changes in testicular weight and serum gonadotropin and testosterone levels before, during, and after birth in the perinatal rat. Endocrinology. 1978;103(6):1985-1991. [DOI] [PubMed] [Google Scholar]

[CIT0016] 16. Clarkson J, Busby ER, Kirilov M, Schütz G, Sherwood NM, Herbison AE. Sexual differentiation of the brain requires perinatal kisspeptin-GnRH neuron signaling. J Neurosci. 2014;34(46):15297-15305. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0017] 17. Slob AK, Ooms MP, Vreeburg JT. Prenatal and early postnatal sex differences in plasma and gonadal testosterone and plasma luteinizing hormone in female and male rats. J Endocrinol. 1980;87(1):81-87. [DOI] [PubMed] [Google Scholar]

[CIT0018] 18. McGivern RF, Hermans RHM, Handa RJ, Longo LD. Plasma testosterone surge and luteinizing hormone beta (LH-b) following parturition: lack of association in the male rat. Eur J Endocrinol. 1995;133(3):366-374. [DOI] [PubMed] [Google Scholar]

[CIT0019] 19. Poling MC, Kauffman AS. Sexually dimorphic testosterone secretion in prenatal and neonatal mice is independent of kisspeptin-Kiss1r and GnRH signaling. Endocrinology. 2012;153(2):782-793. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0020] 20. Kumar D, Freese M, Drexler D, Hermans-Borgmeyer I, Marquardt A, Boehm U. Murine arcuate nucleus kisspeptin neurons communicate with GnRH neurons in utero. J Neurosci. 2014;34(10):3756-3766. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0021] 21. Kumar D, Periasamy V, Freese M, Voigt A, Boehm U. In utero development of kisspeptin/GnRH neural circuitry in male mice. Endocrinology. 2015;156(9):3084-3090. [DOI] [PubMed] [Google Scholar]

[CIT0022] 22. Kauffman AS, Park JH, McPhie-Lalmansingh AA, et al. The kisspeptin receptor GPR54 is required for sexual differentiation of the brain and behavior. J Neurosci. 2007;27(33):8826-8835. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0023] 23. Nakamura S, Watanabe Y, Goto T, Ikegami K, Inoue N, Uenoyama Y, Tsukamura H. Kisspeptin neurons as a key player bridging the endocrine system and sexual behavior in mammals. Front Neuroendocrinol. Published online October 29, 2021. Doi: 10.1016/j.yfrne.2021.100952 [DOI] [PubMed] [Google Scholar]

[CIT0024] 24. Brooks AN, Hagan DM, Sheng C, McNeilly AS, Sweeney T. Prenatal gonadotrophins in the sheep. Anim Reprod Sci. 1996;42(1-4):471-481. [Google Scholar]

[CIT0025] 25. Amodei R, Gribbin K, He W, et al. Role for kisspeptin and neurokinin B in regulation of luteinizing hormone and testosterone secretion in the fetal sheep. Endocrinology. 2020;161(4):1-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0026] 26. Clark SJ, Hauffa BP, Rodens KP, Styne DL, Kaplan SL, Grumbach MM. Hormone ontogeny in the ovine fetus: XIX: The effect of a potent luteinizing hormone-releasing factor agonist on gonadotropin and testosterone release in the fetus and neonate. Pediatr Res. 1989;25(4):347-352. [DOI] [PubMed] [Google Scholar]

[CIT0027] 27. Brooks AN, Currie IS, Gibson F, Thomas GB. Neuroendocrine regulation of sheep fetuses. J Reprod Fertil Suppl. 1992;45:69-84. [PubMed] [Google Scholar]

[CIT0028] 28. Niswender GD, Reichert LE Jr, Midgley AR Jr, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology. 1969;84(5):1166-1173. [DOI] [PubMed] [Google Scholar]

[CIT0029] 29. Dziennis S, Jia T, Rønnekleiv OK, Hurn PD, Alkayed NJ. Role of signal transducer and activator of transcription-3 in estradiol-mediated neuroprotection. J Neurosci. 2007;27(27):7268-7274. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0030] 30.RRID:AB_2619946. https://antibodyregistry.org/search.php?q=AB_2619946

[CIT0031] 31.RRID:AB_2629449. https://antibodyregistry.org/search.php?q=AB_2629449

[CIT0032] 32. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-682. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0033] 33. Roselli CE, Reddy RC, Estill CT, et al. Prenatal influence of an androgen agonist and antagonist on the differentiation of the ovine sexually dimorphic nucleus in male and female lamb fetuses. Endocrinology. 2014;155(12):5000-5010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0034] 34. Jarred RA, Cancilla B, Richards M, Groome NP, McNatty KP, Risbridger GP. Differential localization of inhibin subunit proteins in the ovine testis during fetal gonadal development. Endocrinology. 1999;140(2):979-986. [DOI] [PubMed] [Google Scholar]

[CIT0035] 35. Engelhardt H, Harkness LM, Thomas GB, Brooks AN, McNeilly AS, Baird DT. Expression of inhibin alpha- and beta A-subunit mRNA and protein in the fetal sheep ovary throughout gestation. Mol Cell Endocrinol. 1995;107(2):141-147. [DOI] [PubMed] [Google Scholar]

[CIT0036] 36. Matwijiw I, Faiman C. Control of gonadotropin secretion in the ovine fetus. IV. Male-specific entrainment of the hypothalamic control of luteinizing hormone secretion by testosterone in the female ovine fetus. Endocrinology. 1991;129(3):1447-1451. [DOI] [PubMed] [Google Scholar]

[CIT0037] 37. Sklar CA, Mueller PL, Gluckman PD, Kaplan SL, Rudolph AM, Grumbach MM. Hormone ontogeny in the ovine fetus. VII. Circulating luteinizing hormone and follicle-stimulating hormone in mid- and late gestation. Endocrinology. 1981;108(3):874-880. [DOI] [PubMed] [Google Scholar]

[CIT0038] 38. Foster DL, Roach JF, Karsch FJ, Norton HW, Cook B, Nalbandov AV. Regulation of luteinizing hormone in the fetal and neonatal lamb. I. LH concentrations in blood and pituitary. Endocrinology. 1972;90(1):102-111. [DOI] [PubMed] [Google Scholar]

[CIT0039] 39. Juengel JL, Sawyer HR, Smith PR, et al. Origins of follicular cells and ontogeny of steroidogenesis in ovine fetal ovaries. Mol Cell Endocrinol. 2002;191(1):1-10. [DOI] [PubMed] [Google Scholar]

[CIT0040] 40. Thomas GB, McNeilly AS, Gibson F, Brooks AN. Effects of pituitary-gonadal suppression with a gonadotrophin-releasing hormone agonist on fetal gonadotrophin secretion, fetal gonadal development and maternal steroid secretion in the sheep. J Endocrinol. 1994;141(2):317-324. [DOI] [PubMed] [Google Scholar]

[CIT0041] 41. Connolly F, Rae MT, Bittner L, Hogg K, McNeilly AS, Duncan WC. Excess androgens in utero alters fetal testis development. Endocrinology. 2013;154(5):1921-1933. [DOI] [PubMed] [Google Scholar]

[CIT0042] 42. Roselli CE, Amodei R, Gribbin KP, Corder K, Stormshak F, Estill CT. Excess testosterone exposure alters hypothalamic-pituitary-testicular axis dynamics and gene expression in sheep fetuses. Endocrinology. 2016;157(11):4234-4245. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0043] 43. Taniguchi H, Katano T, Nishida K, Kinoshita H, Matsuda T, Ito S. Elucidation of the mechanism of suppressed steroidogenesis during androgen deprivation therapy of prostate cancer patients using a mouse model. Andrology. 2016;4(5):964-971. [DOI] [PubMed] [Google Scholar]

[CIT0044] 44. Broqua P, Riviere PJ, Conn PM, Rivier JE, Aubert ML, Junien JL. Pharmacological profile of a new, potent, and long-acting gonadotropin-releasing hormone antagonist: degarelix. J Pharmacol Exp Ther. 2002;301(1):95-102. [DOI] [PubMed] [Google Scholar]

[CIT0045] 45. Hannan MA, Kawate N, Fukami Y, et al. Effects of long-acting GnRH antagonist, degarelix acetate, on plasma insulin-like peptide 3, testosterone and luteinizing hormone concentrations, and scrotal circumference in male goats. Theriogenology. 2017;88:228-235. [DOI] [PubMed] [Google Scholar]

[CIT0046] 46. Princivalle M, Broqua P, White R, et al. Rapid suppression of plasma testosterone levels and tumor growth in the dunning rat model treated with degarelix, a new gonadotropin-releasing hormone antagonist. J Pharmacol Exp Ther. 2007;320(3):1113-1118. [DOI] [PubMed] [Google Scholar]

[CIT0047] 47. Kawate N, Kanuki R, Hannan MA, Weerakoon WWPN. Inhibitory effects of long-term repeated treatments of a sustainable GnRH antagonist, degarelix acetate, on caprine testicular functions. J Reprod Dev. 2020;66(6):587-592. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0048] 48. Kittai AS, Blank J, Graff JN. Gonadotropin-releasing hormone antagonists in prostate cancer. Oncology (Williston Park). 2018;32(12):599-602, 604. [PubMed] [Google Scholar]

[CIT0049] 49. Miller DW, Fraser HM, Brooks AN. Suppression of fetal gonadotrophin concentrations by maternal passive immunization to GnRH in sheep. J Reprod Fertil. 1998;113(1):69-73. [DOI] [PubMed] [Google Scholar]

PERMALINK

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses

Rebecka Amodei

Sonnet S Jonker

William Whitler

Charles T Estill

Charles E Roselli

Abstract

Materials and Methods

Animals

Fetal Injections

Fetal Catheterization

GnRH Challenge

Hormone Assays

Tissue Collection

Quantitative Reverse Transcription Real-time Polymerase Chain Reaction

Immunohistochemistry

Microscopy and Image Analysis

Statistics

Results

Effect of Degarelix Treatment on Baseline and GnRH-stimulated LH and T Release

Figure 1.

Figure 2.

Figure 3.

Effect of Degarelix Treatment on Pituitary Gonadotropin Subunit Expression and c-fos Response to GnRH

Figure 4.

Figure 5.

Effect of Degarelix Treatment on the Expression of Steroidogenic Enzymes and LH receptor in the Gonads

Figure 6.

Effect of Degarelix Treatment on Physical Measurements

Table 1.

Discussion

Acknowledgments

Financial Support

Disclosure Statement

Data Availability

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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