In vitro matured oocytes have a higher developmental potential than in vivo matured oocytes after hormonal ovarian stimulation in Callithrix jacchus.

BACKGROUND: The common marmoset, Callithrix jacchus, is an invaluable model in biomedical research. Its use includes genetic engineering applications, which require manipulations of oocytes and production of embryos in vitro. To maximize the recovery of oocytes suitable for embryo production and to fulfil the requirements of the 3R principles to the highest degree possible, optimization of ovarian stimulation protocols is crucial. Here, we compared the efficacy of two hormonal ovarian stimulation approaches: 1) stimulation of follicular growth with hFSH followed by triggering of oocyte maturation with hCG (FSH + hCG) and 2) stimulation with hFSH only (FSH-priming). METHODS: In total, 14 female marmosets were used as oocyte donors in this study. Each animal underwent up to four surgical interventions, with the first three performed as ovum pick-up (OPU) procedures and the last one being an ovariohysterectomy (OvH). In total, 20 experiments were carried out with FSH + hCG stimulation and 18 with FSH-priming. Efficacy of each stimulation protocol was assessed through in vitro maturation (IVM), in vitro fertilization (IVF) and embryo production rates. RESULTS: Each study group consisted of two subgroups: the in vivo matured oocytes and the oocytes that underwent IVM. Surprisingly, in the absence of hCG triggering some of the oocytes recovered were at the MII stage, moreover, their number was not significantly lower compared to FSH + hCG stimulation (2.8 vs. 3.9, respectively (ns)). While the IVM and IVF rates did not differ between the two stimulation groups, the IVF rates of in vivo matured oocytes were significantly lower compared to in vitro matured ones in both FSH-priming and FSH + hCG groups. In total, 1.7 eight-cell embryos/experiment (OPU) and 2.1 eight-cell embryos/experiment (OvH) were obtained after FSH + hCG stimulation vs. 1.8 eight-cell embryos/experiment (OPU) and 5.0 eight-cell embryos/experiment (OvH) following FSH-priming. These numbers include embryos obtained from both in vivo and in vitro matured oocytes. CONCLUSION: A significantly lower developmental competence of the in vivo matured oocytes renders triggering of the in vivo maturation with hCG as a part of the currently used FSH-stimulation protocol unnecessary. In actual numbers, between 1 and 7 blastocysts were obtained following each FSH-priming. In the absence of further studies, FSH-priming appears superior to FSH + hCG stimulation in the common marmoset under current experimental settings.

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.

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.

Developmental and family history-based analysis of congenital fused labia phenotype in the captive common marmoset (Callithrix jacchus).

BACKGROUND: Congenital fused labia (CFL) is defined as a failure or significant delay in the opening of the juvenile sealed labia majora. This phenotype is known to be variably common in adult captive female marmosets but has never been investigated in detail before. MATERIALS AND METHODS: Here, we define, describe and quantify the variations in the degree of closure of the vulva in 122 captive marmosets (Callithrix jacchus) from 1.2 to 42 months old and include colony analysis. RESULTS: There was a negative correlation between the degree of labial fusion and animal age after prepubertal period (P < 0.05). CFL females had higher number CFL relatives (4.3 ± 0.6 vs 2.4 ± 0.5 for non-CFL, P < 0.05) and more external ancestors compared to non-CFL (P < 0.05). CONCLUSIONS: Our results therefore suggest that CFL phenotype is most likely associated with epigenetic effects induced by the captive environment and colony management strategy of extensive crossing of family lines to promote heterozygosity.

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.

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.