Restoration of testis function in hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by pharmacoperone drug therapy.

Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.

A Comparison of Oocyte Yield between Ultrasound-Guided and Laparoscopic Oocyte Retrieval in Rhesus Macaques.

Obtaining quality oocytes is a prerequisite for ART-based studies. Here we describe a method for transabdominal ultrasound-guided (US) oocyte retrieval in rhesus macaques (Macaca mullata) and compare it to the standard surgical approach using laparoscopy (LAP). We analyzed oocyte yield from six continuous reproductive seasons (2017-2023) that included n = 177 US-guided and n = 136 laparoscopic oocyte retrievals. While the ultrasound-guided technique retrieved significantly fewer oocytes on average (LAP: 40 ± 2 vs. US: 27 ± 1), there was no difference in the number of mature metaphase II oocytes (MII) between the two techniques (LAP: 17 ± 1 vs. US: 15 ± 1). We show that oocytes retrieved by the ultrasound-guided approach fertilize at the same rates as those obtained via the laparoscopic procedure (LAP Fert Rate: 84% ± 2% vs. US Fert Rate: 83% ± 2%). In conclusion, minimally invasive ultrasound-guided oocyte retrieval improves animal welfare while delivering equivalent numbers of mature oocytes, which are ideal for ART. Furthermore, we show that oocyte competency, as represented by fertilization rate, is not affected by retrieval technique. Therefore, the Oregon National Primate Research Center (ONPRC) has adopted the ultrasound-guided approach as the standard technique for oocyte retrieval.

In vivo delivery of FTY720 prevents radiation-induced ovarian failure and infertility in adult female nonhuman primates.

OBJECTIVE: To determine whether sphingosine-1-phosphate (S1P), or the S1P mimetic FTY720 shields ovaries of adult female rhesus monkeys from damage caused by 15 Gy of targeted radiotherapy, allowing for the retention of long-term fertility, and to evaluate whether S1P protects human ovarian tissue (xenografted into mice) from radiation-induced damage. DESIGN: Research animal study. SETTING: Research laboratory and teaching hospital. PATIENT(S): Adult female rhesus macaques (8-14 years of age; n = 21) and two women (24 and 27 years of age) undergoing gynecologic surgery for benign reasons, after informed consent and approval. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian histologic analysis, ovarian reserve measurements, and fertility in mating trials. RESULT(S): Rapid ovarian failure was induced in female macaques by ovarian application of 15 Gy of radiation. Females given S1P or FTY720 by direct intraovarian cannulation for 1 week before ovarian irradiation rapidly resumed menstrual cycles because of maintenance of follicles, with greater beneficial effects achieved using FTY720. Monkeys given the S1P mimetic before ovarian irradiation also became pregnant in mating trials. Offspring conceived and delivered by radioprotected females developed normally and showed no evidence of genomic instability, as measured by micronucleus frequency in reticulocytes. Adult human ovarian cortical tissue xenografted into mice also exhibited a reduction in radiation-induced primordial oocyte depletion when preexposed to S1P. CONCLUSION(S): S1P and its analogs hold clinical promise as therapeutic agents to preserve ovarian function and fertility in female cancer patients exposed to cytotoxic treatments.

Use of controlled ovulation of the dominant follicle to assess oocyte maturation during natural menstrual cycles in rhesus macaques.

To determine the practicality of controlled ovulation of the dominant follicle as a technique to study meiotic maturation of oocytes during contraceptive research, we developed a technique for aspiration of the single dominant follicle using a dual-needle continuous irrigation technique 27 hours after an ovulatory stimulus. All of the oocytes (3/3) recovered from control animals, but only 1/6 (17%) of oocytes from animals treated with the meiotic inhibitor ORG 9935 exhibited germinal vesicle breakdown, indicating resumption of meiosis.