Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library

Ovulation-selective/specific genes, that is, genes preferentially or exclusively expressed during the ovulatory process, have been the subject of growing interest. We report herein studies on the use of suppression subtractive hybridization (SSH) to construct a 'forward' ovulation-selective/specific cDNA library. In toto, 485 clones were sequenced and analyzed for homology to known genes with the basic local alignment tool (BLAST). Of those, 252 were determined to be nonredundant. Of these 252 nonredundant clones, 98 were analyzed by probing mouse preovulatory and postovulatory ovarian cDNA. Twenty-five clones (26%) failed to show any signal, and 43 cDNAs tested thus far display a true ovulation-selective/specific expression pattern. In this communication, we focus on one such ovulation-selective gene, the fatty acid elongase 1 (FAE-1) homolog, found to be localized to the inner periantral granulosa and to the cumulus granulosa cells of antral follicles. The FAE-1 gene is a beta-ketoacyl-CoA synthase belonging to the fatty acid elongase (ELO) family, which catalyzes the initial step of very long-chain fatty acid synthesis. All in all, the present study accomplished systematic identification of those hormonally regulated genes that are expressed in the ovary in an ovulation-selective/specific manner. These ovulation-selective/specific genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for both the promotion of fertility and its control.     

Discovery of LH-regulated genes in the primate corpus luteum

Circulating LH is essential for the development and function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular and molecular processes whereby LH controls luteal structure and function are poorly understood. Therefore, studies were initiated to identify gene products that are regulated by gonadotrophin in the monkey CL. Rhesus monkeys either were untreated (controls, CTRL; n = 3) or received the GnRH antagonist Antide (ANT; 3 mg/kg body weight, n = 3) to inhibit pituitary LH secretion on day 6 of the luteal phase in spontaneous menstrual cycles. The CL was removed 24 h later. RNA was extracted and converted to cDNA. The CTRL and ANT cDNA were differentially labelled with fluorescent dyes (Cy3-CTRL and Cy5-ANT) and hybridized onto microarrays containing 11,600 human cDNA. The selected cDNA were analysed further via semi-quantitative RT-PCR (a) to validate the microarray results and (b) to determine if their expression varies in the CL (n = 3/stage) between the mid (day 6-8), late (day 14-16), or very late (day 18-19, menses) luteal phase of the natural cycle. After normalization of the fluorescence data, 206 cDNA (1.8% of the total) exhibited > or = 2-fold change in expression after ANT. Of the 25 cDNA exhibiting a > or = 6-fold change, 6 were up-regulated and 19 were down-regulated. Twenty-two of these 25 cDNA were validated by RT-PCR as differentially expressed in the ANT group, relative to the CTRL group, and 11 of 25 changed (P < 0.05) correspondingly in the late-to-very late luteal phase. Thus, we have identified gene products that are regulated by gonadotrophin in the primate CL that may be important in luteal regression during the menstrual cycle.     

Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Nonhuman primates (NHPs) are critical for biomedical research due to their close genetic and physiologic relationship to humans. In addition, according to the classification criteria proposed by the International Union for the Conservation of Nature, approximately 63% of the 702 species and subspecies of NHP in the world are at some level of risk of extinction.^21,31 Thus, efficient assisted reproductive technologies (ARTs) are required to 1) understand primate reproductive processes, 2) advance NHP biomedical model development and 3) advance species conservation efforts.The rhesus macaque (Macaca mulatta) is one of the most commonly used NHP species for research studies relevant to human health and disease.⁴² Advances in the use of ARTs in this important species include cryopreservation of sperm;⁴¹ artificial insemination;³⁴ in vitro fertilization (IVF);³ and intracytoplasmic sperm injection (ICSI).³⁰ Establishing effective and reproducible rhesus macaque ARTs is critical in allowing the birth of live offspring after blastomere nuclear transfer,²⁸ somatic cell nuclear transfer,¹⁰ mitochondrial replacement in oocytes,⁴³ and autologous grafting of prepubertal rhesus testis, followed by ICSI and embryo development.¹⁶ Moreover, recent advances in gene editing now allow NHP genome manipulation in one cell embryos (zygotes) for the subsequent generation of transgenic models of human disease.¹¹Genetic resource banking or cryobanking, (the low temperature storage of gametes, embryos, and tissues) is crucial to preserving genetic material for future use in ARTs. Cryobanking allows the preservation of material from genetically valuable individuals independent of the long-term goal for its use (biomedical models, domestic species breeding programs, species conservation, etc.).⁶ Moreover, cryobanking makes sperm readily available when needed, without the need to maintain live male sperm donors or personnel who are trained to collect fresh sperm.However, spermatozoa cryopreservation and recovery of viable sperm after thawing is still a major concern in NHP ARTs and would benefit from further improvement.⁵⁰ Although several studies have investigated the optimal conditions for primate sperm preservation,^{12-14,25,33,39-41,53,54} major gaps exist in our understanding of what is critical for promoting the survival of sperm after thawing. For example, IVF success using fresh sperm varied between 50% to 61%^50,51 with 55% of the embryos reaching the blastocyst stage,⁵¹ whereas IVF with cryopreserved sperm resulted in 82 ± 13% of the ova developing into embryos, of which only 39 ± 21% reached the blastocyst stage.⁴¹ One process that needs further optimization in rhesus macaques is sperm vitrification. Vitrification is the process of obtaining a glass-like solid to avoid ice crystal formation; it is achieved either by rapid cooling or using additives.⁵² Although successful vitrification of human sperm has been reported,^19,20 the only study that assessed sperm vitrification in rhesus macaques reported no motility after thawing.²⁹ Therefore, further studies are necessary on how to successfully cryopreserve sperm from rhesus macaques.An important factor to consider with regard to sperm cryopreservation is how to isolate good quality sperm after thawing (that is, progressively motile sperm with an intact plasma membrane and acrosome and low levels of DNA fragmentation). Several studies have compared different sperm preparation methods in various species, including bulls,^2,23 humans,¹⁵ and dogs,²² but no studies to date have systematically compared various methods in rhesus macaques.Therefore, the objectives of the current study were to 1) compare 2 methods for cryopreservation of sperm from rhesus macaques with regard to sperm viability characteristics after thawing; 2) compare 4 different methods of removal of cryopreservation reagents and sperm isolation after thawing, and (3) evaluate the overall efficacy of the 2 best sperm isolation methods for use in IVF.     

Systematic analysis of protease gene expression in the rhesus macaque ovulatory follicle: metalloproteinase involvement in follicle rupture

Protease genes were identified that exhibited increased mRNA levels before and immediately after rupture of the naturally selected, dominant follicle of rhesus macaques at specific intervals after an ovulatory stimulus. Quantitative real-time PCR validation revealed increased mRNA levels for matrix metalloproteinase (MMP1, MMP9, MMP10, and MMP19) and a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS1, ADAMTS4, ADAMTS9, and ADAMTS15) family members, the cysteine protease cathepsin L (CTSL), the serine protease urokinase-type plasminogen activator (PLAU), and the aspartic acid protease pepsinogen 5 (PGA5). With the exception of MMP9, ADAMTS1, and PGA5, mRNA levels for all other up-regulated proteases increased significantly (P < 0.05) 12 h after an ovulatory human chorionic gonadotropin (hCG) bolus. MMP1, -10, and -19; ADAMTS1, -4, and -9; CTSL; PLAU; and PGA5 also exhibited a secondary increase in mRNA levels in 36-h postovulatory follicles. To further determine metalloproteinase involvement in ovulation, vehicle (n = 4) or metalloproteinase inhibitor (GM6001, 0.5 μg/follicle, n = 8) was injected into the preovulatory follicle at the time of hCG administration. Of the eight GM6001-injected follicles, none displayed typical stigmata indicative of ovulation at 72 h after hCG; whereas all four vehicle-injected follicles ovulated. No significant differences in mean luteal progesterone levels or luteal phase length occurred between the two groups. Subsequent histological analysis revealed that vehicle-injected follicles ruptured, whereas GM6001-injected follicles did not, as evidenced by an intact stroma and trapped oocytes (n = 3). These findings demonstrate metalloproteinases are critical for follicle rupture in primates, and blocking their activity would serve as a novel, nonhormonal means to achieve contraception.     

Oocyte maturation and in vitro hormone production in small antral follicles (SAFs) isolated from rhesus monkeys

Purpose

The small antral follicles (SAFs) from the ovarian medulla can be a potential source of oocytes for infertility patients, but little is known about their ability to yield mature oocytes. This study evaluated the response of these SAFs to a stimulatory bolus of human corionic gonadotropin (hCG) in vitro.

Methods

Oocyte nuclear maturation and hormone production (estradiol [E2], progesterone [P4]), antimullerian hormone [AMH]) by individual intact SAFs (n = 91; >0.5 mm; n = 5 monkeys) was evaluated after 34 h of culture in the absence (control) or presence of hCG.

Results

Of the total cohort (n = 91), 49 % of SAFs contained degenerating oocytes. The percentage of healthy oocytes able to reinitiate meiosis to the metaphase I (MI) and MII was greater (p < 0.05) after hCG compared to controls. E2, P4 and AMH levels were higher (p < 0.05) in SAF cultures containing germinal vesicle (GV) oocytes compared to those with MII oocytes regardless of hCG exposure. SAF with MI oocytes produced more E2, but less (p < 0.05) P4 and AMH compared to SAFs containing GV oocytes (p < 0.05). Follicles ≥1 mm produced more (p < 0.05) E2, whereas follicle diameter did not correlate with P4 or AMH levels. Only P4 increased (p < 0.05) in response to hCG, regardless of follicle size or oocyte maturity. SAFs containing degenerating oocytes produced similar levels of E2, P4 and AMH compared to SAFs containing healthy oocytes.

Conclusions

These data indicate, for the first time, that oocytes within primate SAFs can reinitiate meiosis in vitro in the absence of hCG, but nuclear maturation is enhanced in SAFs cultured with hCG. Oocyte nuclear maturation within SAFs in is associated with decreased E2, P4 and AMH levels. Furthermore, hormone content within the culture media does not necessarily reflect oocyte quality.     

Inhibition of skin 11β-hydroxysteroid dehydrogenase activity in vivo potentiates the anti-inflammatory actions of glucocorticoids

Abstract Synthetic forms of glucocorticoids (GCs) with high potency are widely used to treat a number of dermatological conditions having an inflammatory or autoimmune etiology. While GCs are generally effective in their ability to suppress inflammatory processes, their chronic use can lead to detrimental systemic side effects. In this report, we describe a method by which the localized antiinflammatory potential of the natural GC cortisol can be significantly augmented without increasing the risk of negative systemic effects. 11β-Hydroxysteroid dehydrogenase (11β-HSD) is a naturally occurring enzyme in the skin. 11β-HSD functionally converts biologically active 11-hydroxy GCs to their biologically inactive 11-keto metabolites, thereby limiting the ability of GCs to mediate antiinflammatory activities. By topically applying specific inhibitors of 11β-HSD in conjunction with low doses of GCs, the antiinflammatory properties of cortisol can be significantly potentiated. It was observed that the generation of the effector phase of contact hypersensitivity (CH) responses could be suppressed by this combined treatment under conditions where the 11β-HSD inhibitor alone, or cortisol alone were only minimally effective. Only the combined treatment was effective at inhibiting the progression of an ongoing CH response. Received: 22 July 1997     

Characterization of the ovarian transcriptome through the use of differential analysis of gene expression methodologies

Prior to the development of high-throughput methods for the analysis of differential gene expression, genes required for proper ovarian function were identified on a case-by-case basis. Recently, however, several techniques have been developed that enable investigators to study large-scale changes in gene expression under a variety of experimental conditions. The utilization of these methodologies has led to the identification of a number of novel or previously unappreciated genes that are expressed within distinct cell types in the ovary or at specific stages of the ovarian cycle. This review details the recent use of differential analysis strategies in identifying (i) genes that are expressed exclusively or preferentially in the ovary, (ii) genes that are differentially expressed in isolated ovarian cells in response to hormonal stimulation, and (iii) those genes that are expressed at specific stages of the ovarian cycle. The genes identified through the use of these approaches represent potential targets for designing agents capable of regulating ovarian physiology and thus fertility.