Microtubule-Targeting Agents: Disruption of the Cellular Cytoskeleton as a Backbone of Ovarian Cancer Therapy.

Microtubules are dynamic polymers composed of α- and ß-tubulin heterodimers. Microtubules are universally conserved among eukaryotes and participate in nearly every cellular process, including intracellular trafficking, replication, polarity, cytoskeletal shape, and motility. Due to their fundamental role in mitosis, they represent a classic target of anti-cancer therapy. Microtubule-stabilizing agents currently constitute a component of the most effective regimens for ovarian cancer therapy in both primary and recurrent settings. Unfortunately, the development of resistance continues to present a therapeutic challenge. An understanding of the underlying mechanisms of resistance to microtubule-active agents may facilitate the development of novel and improved approaches to this disease.

Tubulin Complexity in Cancer and Metastasis.

Tubulin plays a fundamental role in cellular function and as the subject for microtubule-active agents in the treatment of ovarian cancer. Microtubule-binding proteins (e.g., tau, MAP1/2/4, EB1, CLIP, TOG, survivin, stathmin) and posttranslational modifications (e.g., tyrosination, deglutamylation, acetylation, glycation, phosphorylation, polyamination) further diversify tubulin functionality and may permit additional opportunities to understand microtubule behavior in disease and to develop microtubule-modifying approaches to combat ovarian cancer. Tubulin-based structures that project from suspended ovarian cancer cells known as microtentacles may contribute to metastatic potential of ovarian cancer cells and could represent an exciting novel therapeutic target.

Mechanistic insight into efficient H2 generation upon HCOONa hydrolysis.

Sodium formate (SF) is regarded as a technological additive to improve H2 generation upon HCOOH dehydrogenation. The development of SF as a hydrogen storage material is still a challenge. Herein, we report the design and synthesis of carbon nanosphere-stabilized Pd nanoparticles (Pd/CNS) for the direct H2 generation upon SF hydrolysis in the presence of Fe3+. The tandem reaction, isotopic mass spectrometry, and gas chromatograms result confirmed that SF hydrolysis generates H2 with one H atom provided by SF and other H by H2O. The kinetic study and detailed mechanistic investigations have demonstrated that the concerted process between the cleavage of O-H bond in H2O and -O2C-H bond oxidative addition is the rate-controlling step in SF hydrolysis. This work offers a new chemical hydrogen storage material (HCOONa) for the high-efficiency generation, transport, and storage of H2.

"On-Off" Control for On-Demand H2 Release upon Dimethylamineborane Hydrolysis over Ru0.8Ni0.2/MoS2 Nanohybrids.

In spite of the fact that remarkable developments are achieved in the design and development of novel nanocatalysts for H2 release upon dimethylamineborane hydrolysis, the development of an "on-off" switch for demand-based H2 evolution upon dimethylamineborane hydrolysis is still a matter of supreme importance, however. Herein, we synthesized a string of MoS2 nanosheet-supported RuNi bimetallic nanohybrids (RuxNi1-x/MoS2), by fixation of RuNi nanoparticles at the MoS2 surface, for the H2 evolution upon the hydrolysis of dimethylamineborane at 30 °C. For safely and effectively generating, transporting, and storing H2 gas, the selective "on-off" switch for on-demand H2 evolution upon dimethylamineborane hydrolysis over the Ru0.8Ni0.2/MoS2 nanohybrid has been successfully realized by the Zn2+/EDTA-2Na system. In particular, the H2 evolution is totally switched off by adding Zn(NO3)2. It seems that Zn2+ ions are attached and anchored at the Ru0.8Ni0.2/MoS2 surface, inhibiting their surface-active sites, leading to the termination of H2 evolution. Then, the H2 generation is subsequently reactivated by adding the EDTA-2Na solution because of its excellent coordination ability with Zn2+ ions. This study not only offers a new and efficient RuNi nanocatalyst for dimethylamineborane hydrolysis but also proposes a new method for the demand-based H2 production.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

A brain-specific pgc1α fusion transcript affects gene expression and behavioural outcomes in mice.

PGC1α is a transcriptional coactivator in peripheral tissues, but its function in the brain remains poorly understood. Various brain-specific Pgc1α isoforms have been reported in mice and humans, including two fusion transcripts (FTs) with non-coding repetitive sequences, but their function is unknown. The FTs initiate at a simple sequence repeat locus ∼570 Kb upstream from the reference promoter; one also includes a portion of a short interspersed nuclear element (SINE). Using publicly available genomics data, here we show that the SINE FT is the predominant form of Pgc1α in neurons. Furthermore, mutation of the SINE in mice leads to altered behavioural phenotypes and significant up-regulation of genes in the female, but not male, cerebellum. Surprisingly, these genes are largely involved in neurotransmission, having poor association with the classical mitochondrial or antioxidant programs. These data expand our knowledge on the role of Pgc1α in neuronal physiology and suggest that different isoforms may have distinct functions. They also highlight the need for further studies before modulating levels of Pgc1α in the brain for therapeutic purposes.

Vitamin D in follicular development and oocyte maturation.

Vitamin D (VD) is a secosteroid hormone synthesized predominantly in the skin upon UV light exposure, which can also be obtained from dietary sources. In target cells, the bioactive VD binds to specific VD receptor to regulate downstream transcription of genes that are involved in a wide range of cellular processes. There is an increasing recognition that the proper physiological levels of VD are critical for optimizing reproductive potential in women. The direct VD action in the ovary was first suggested in the 1980s. Since then, research has attempted to determine the role of VD in follicular development and oocyte maturation in animal models and clinical settings. However, data published to date are inconclusive due to the complexity in VD metabolism and the fact that VD actions are pervasive in regulating physiological functions in various systems, including the reproductive, endocrine and nervous systems that control reproduction. This review summaries in vitro, in vivo, and clinical evidence regarding VD metabolism and signaling in the ovary, as well as VD-regulated or VD-associated ovarian follicular development, steroidogenic function, and oocyte maturation. It is suggested that adequate animal models are needed for well-controlled studies to unravel molecular mechanisms of VD action in the ovary. For clinical studies, follicular development and function may be evaluated more effectively in a relatively homogeneous patient population under a well-controlled experimental design. A comprehensive understanding of VD-regulated folliculogenesis and oogenesis will provide critical insight into the impact of VD in female reproductive health.

Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

STUDY QUESTION: Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? SUMMARY ANSWER: In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. WHAT IS KNOWN ALREADY: Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. STUDY DESIGN, SIZE, DURATION: Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. LIMITATIONS, REASONS FOR CAUTION: Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. WIDER IMPLICATIONS OF THE FINDINGS: Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. STUDY FUNDING/COMPETING INTEREST(S): NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Anti-Müllerian Hormone Regulation of Synaptic Transmission in the Hippocampus Requires MAPK Signaling and Kv4.2 Potassium Channel Activity.

Anti-Müllerian hormone (AMH) is a paracrine factor generated peripherally by the gonads to regulate gonadal function in adult mammals. We recently reported that AMH and AMH-specific receptor Anti-Müllerian hormone receptor 2 (AMHR2) are expressed in the hippocampus, and exogenous AMH protein rapidly increased synaptic transmission and long-term synaptic plasticity at the CA3-CA1 synapses. Here we examined the cell-specific expression of AMHR2 and the cellular mechanism of rapid boosting effect of AMH on synaptic transmission in mouse hippocampus. Immunofluorescence staining showed that AMHR2 was specifically expressed in the soma and dendrites of hippocampal pyramidal neurons, but not glial cells. Electrophysiological recordings on acute hippocampal slices showed that AMH did not affect AMPAR-mediated or N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents at the CA3-CA1 synapses. The small-conductance Ca2+-activated K+ channel (SK2) and A-type K+ channel (Kv4.2) contribute to shaping excitatory postsynaptic potentials (EPSPs) at the CA3-CA1 synapses. Bath application of apamin to block SK2 did not alter AMH effect on increasing EPSPs, whereas blocking Kv4.2 channel with 4-aminopyridine, or chelating internal Ca2+ with BAPTA occluded the action of AMH on boosting EPSPs. Kv4.2 activity is regulated by p38 mitogen-activated kinase (MAPK). Blocking p38 MAPK with SB203580 occluded the effect of AMH on increasing EPSPs. These results show that Kv4.2 channel contributes to the rapid action of AMH on boosting synaptic transmission in a Ca2+- and p38 MAPK-dependent manner. Our findings provide functional evidence that AMH enhances synaptic transmission through Kv4.2 channel in the hippocampus, suggesting a possible role of Kv4.2 channel in AMH-regulated neuronal process underlying learning and memory.

Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Association between vitamin D and ovarian cancer development in BRCA1 mutation carriers.

OBJECTIVE: Women with inherited mutations in BRCA1 gene have a high (40-70%) genetic risk of developing ovarian cancer. Epidemiological studies suggest an inverse correlation between serum vitamin D (VD) levels and the risk of ovarian cancer, but there is a lack of data from BRCA1 mutation (BRCA1 mut) carriers. Therefore, we investigated VD levels and actions in cancer free women with BRCA1 mutations. MATERIALS AND METHODS: Blood, ovary and fallopian tube samples were collected from healthy pre-menopausal women with BRCA1 mut and without BRCA1 mutations (BRCA wt). Serum calcifediol (major circulating form of VD) concentrations were measured by electrochemiluminescence immunoassay. Immunohistochemistry was performed on paraffin-embedded ovarian and fallopian tube sections to determine vitamin D receptor (VDR) expression. Ovarian surface epithelial cells (OSEs) from BRCA1 mut carriers were cultured with or without calcitriol supplementation for 72 hrs. VDR protein levels, cell proliferation and cell viability were analyzed. RESULTS: BRCA1 mut women had lower serum calcifediol levels compared to BRCA wt women (p = 0.003). VDR protein expression was evident in ovarian and the fallopian tube epithelium of BRCA wt patients, but was reduced in BRCA1 mut women. Calcitriol (biologically active VD) supplementation elevated VDR expression in cultured BRCA1 mut OSEs (p = 0.005) and decreased cell proliferation rates in a dose-dependent manner without inducing apoptosis. CONCLUSIONS: VD biosynthesis and signaling via VDR in the ovarian and fallopian tube epithelium are impaired in BRCA1 mut women. VD treatment may limit BRCA1 mut epithelial cell proliferation without affecting cell viability, providing a rationale for exploring the potential for VD in ovarian cancer prevention in BRCA1 mut carriers.

Single Nucleotide Resolution Analysis Reveals Pervasive, Long-Lasting DNA Methylation Changes by Developmental Exposure to a Mitochondrial Toxicant.

Mitochondrial-driven alterations of the epigenome have been reported, but whether they are relevant at the organismal level remains unknown. The viable yellow agouti mouse (Avy) is a powerful epigenetic biosensor model that reports on the DNA methylation status of the Avy locus, which is established prior to the three-germ-layer separation, through the coat color of the animals. Here we show that maternal exposure to rotenone, a potent mitochondrial complex I inhibitor, not only changes the DNA methylation status of the Avy locus in the skin but broadly affects the liver DNA methylome of the offspring. These effects are accompanied by altered gene expression programs that persist throughout life, and which associate with impairment of antioxidant activity and mitochondrial function in aged animals. These pervasive and lasting genomic effects suggest a putative role for mitochondria in regulating life-long gene expression programs through developmental nuclear epigenetic remodeling.

Rapid actions of anti-Müllerian hormone in regulating synaptic transmission and long-term synaptic plasticity in the hippocampus.

Anti-Müllerian hormone (Amh) is a peptide factor that is known to regulate sexual differentiation and gonadal function in mammals. Although Amh is also suggested to be associated with cognitive development and function in the postnatal brain, little is known about its expression or direct effects on neuronal activities in the hippocampus. Therefore, we assessed Amh and its receptor expression in the hippocampus of male and female mice using PCR, Western blot, and immunofluorescence staining. While Amh-specific receptor expression was comparable between males and females, mRNA and protein levels of Amh were higher in females than those of males. Electrophysiological recordings on acute hippocampal slices showed that exogenous Amh protein addition increased synaptic transmission and long-term synaptic plasticity at the Cornu Ammonis (CA) 3-CA1 synapses. Amh exposure also increased the excitatory postsynaptic potential at CA1 synapses. Our findings support direct and rapid actions of Amh as a paracrine and/or autocrine factor in regulating hippocampal neuronal activities. Data provide functional evidence of Amh-mediated postsynaptic modulation of synaptic transmission and Amh-regulated long-term synaptic plasticity in the hippocampus. These results suggest a potential role of Amh in learning and memory, and a possible cause of the sex differences in cognitive development and function.

Stage-dependent actions of antimüllerian hormone in regulating granulosa cell proliferation and follicular function in the primate ovary.

OBJECTIVE: To study the direct action and physiological role of antimüllerian hormone (AMH) in regulating ovarian follicular development and function in vivo in primates. DESIGN: Animals were assigned to six treatment sequences in a crossover design study. Intraovarian infusion was performed during the follicular phase of the menstrual cycle with agents including: control vehicle; recombinant human AMH (rhAMH); and neutralizing anti-human AMH antibody (AMHAb). Before ovariectomy after the final treatment, the animals received intravenous injections of bromodeoxyuridine (BrdU). SETTING: National primate research center. ANIMALS: Adult female rhesus macaques (Macaca mulatta). INTERVENTIONS: None. MAIN OUTCOME MEASURES: Cycle length, follicle cohorts, and serum steroid levels were assessed. Ovarian histology, as well as granulosa cell (GC) proliferation and oocyte viability, were evaluated. RESULTS: In vehicle-infused ovaries, a dominant follicle was observed at midcycle E2 peak. However, rhAMH-treated ovaries exhibited an increased number of small antral follicles, whereas AMHAb-treated ovaries developed multiple large antral follicles. Serum E2 levels in the follicular phase decreased after rhAMH infusion and increased after AMHAb infusion. The rhAMH infusion increased serum T levels. Whereas early-growing follicles of rhAMH-treated ovaries contained BrdU-positive GCs, antral follicles containing BrdU-positive GCs were identified in AMHAb-treated ovaries. Autophagy was observed in oocytes of early-growing and antral follicles exposed to AMHAb and rhAMH, respectively. CONCLUSIONS: AMH enhanced early-stage follicle growth, but prevented antral follicle development and function via its stage-dependent regulation of GC proliferation and oocyte viability. This study provides information relevant to the pathophysiology of ovarian dysfunction and the treatment of infertility.

The dilemma of early preventive oophorectomy in familial small cell carcinoma of the ovary of hypercalcemic type.

•New pathogenic familial SMARCA4 variant, c.3081+1G>T.•Prophylactic surgery in healthy carrier of germline SMARCA4 mutation.•Long term hormone therapy in a 13-year-old girl.

Vitamin D3 Regulates Follicular Development and Intrafollicular Vitamin D Biosynthesis and Signaling in the Primate Ovary.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.

Stage-specific modulation of antimüllerian hormone promotes primate follicular development and oocyte maturation in the matrix-free three-dimensional culture.

OBJECTIVE: To study whether follicular growth and oocyte maturation can be improved by antimüllerian hormone (AMH) modulation at specific stages of follicular development. DESIGN: Primary and secondary follicles were cultured in a matrix-free system and were assigned to the control group and the group with AMH supplementation during the preantral stage and neutralizing AMH antibody addition during the antral stage. SETTING: National primate research center. ANIMAL(S): Adult, female rhesus macaques (Macaca mulatta). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle survival, growth, steroid and paracrine factor production, and oocyte competence were evaluated. Follicles were assessed for expression of genes that are critical for gonadotropin signaling, cumulus cell glycolysis, and oocyte quality. RESULT(S): Primary follicles formed "organoids" and developed to the antral stage in group culture. AMH exposure during the preantral stage increased organoid diameters. Oocytes from the AMH-treated organoids had greater diameters and matured to the metaphase II (MII) stage. Secondary follicles developed to the antral stage during individual culture. The AMH exposure during the preantral stage and AMH antibody treatment during the antral stage increased follicle diameters, vascular endothelial growth factor and follistatin production, differentiation factor 9 expression, and oocyte diameters. The MII oocytes from the AMH-modulated group developed to the morula stage after IVF, with one to the blastocyst stage. CONCLUSION(S): AMH supplementation at the preantral stage and depletion at the antral stage enhanced primate follicular development and oocyte competence in vitro. The improved embryonic development supports in vitro follicle maturation as a potential approach for fertility preservation.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Subcutaneous ovarian tissue transplantation in nonhuman primates: duration of endocrine function and normalcy of subsequent offspring as demonstrated by reproductive competence, oocyte production, and telomere length.

PURPOSE: The main purposes of the study were to investigate the endocrine function of ovarian tissue transplanted to heterotopic subcutaneous sites and the reproductive competence and telomere length of a nonhuman primate originating from transplanted tissue. METHODS: Ovarian cortex pieces were transplanted into the original rhesus macaques in the arm subcutaneously, in the abdomen next to muscles, or in the kidney. Serum estradiol (E2) and progesterone (P4) concentrations were measured weekly for up to 8 years following tissue transplantation. A monkey derived from an oocyte in transplanted ovarian tissue entered time-mated breeding and underwent controlled ovarian stimulation. Pregnancy and offspring were evaluated. Telomere lengths and oocytes obtained following controlled ovarian stimulation were assessed. RESULTS: Monkeys with transplants in the arm and abdomen had cyclic E2 of 100 pg/ml, while an animal with arm transplants had E2 of 50 pg/ml. One monkey with transplants in the abdomen and kidney had ovulatory cycles for 3 years. A monkey derived from an oocyte in transplanted tissue conceived and had a normal gestation until intrapartum fetal demise. She conceived again and delivered a healthy offspring at term. Controlled ovarian stimulations of this monkey yielded mature oocytes comparable to controls. Her telomere length was long relative to controls. CONCLUSIONS: Heterotopic ovarian tissue transplants yielded long-term endocrine function in macaques. A monkey derived from an oocyte in transplanted tissue was reproductively competent. Her telomere length did not show epigenetically induced premature cellular aging. Ovarian tissue transplantation to heterotopic sites for fertility preservation should move forward cautiously, yet optimistically.