Graafian Follicle

Fixed ovaries were processed for microscopy and subsequently the entire ovary was sectioned at 8 µm. Every 5^th ovarian section was stained with haematoxylin and eosin for morphometric analysis. In order to prevent multiple counts of the same follicle, only those follicles with a visible oocyte nucleus were included. Since oocyte nuclei measured between 20–30 µm in diameter, counting every 5th section of the ovary ensured a distance of 40 µm between analysed sections, preventing multiple counts of the same ovarian follicle. Follicle classification based on characteristics proposed by Hirshfield & Midgley [66] (link) was as follows: type 1: primordial follicle, one layer of flattened granulosa cells surrounding the oocyte; type 2: primary follicle, one to fewer than two complete layers of cuboidal granulosa cells; type 3: secondary follicle, an oocyte surrounded by greater than one layer of cuboidal granulosa cells, with no visible antrum; type 4: antral follicle, an oocyte surrounded by multiple layers of cuboidal granulosa cells and containing one or more antral spaces, cumulus oophorus and theca layer may also have been evident. Total volume of each section was calculated (area of the section x thickness of the section) and follicle counts for each animal were corrected for the total volume of ovarian tissue counted. All follicle counts were then expressed as number of follicles counted per mm³ of ovarian tissue counted.

Duplicate 10 g samples of each tissue were grounded in liquid nitrogen with mortar and pestle. From the ovary of POST heifers, this included tissue composing antral follicles and the corpus luteum, whereas from PRE heifers, this only included tissue containing antral follicles. Total RNA was purified using a Trizol protocol (Invitrogen, Carlsbad, CA). Quality was evaluated using the RNA Integrity Number (RIN) value from the Experion automated electrophoresis system (BioRad, Hercules, CA). The RIN values ranged from 7.6 to 9.8 in the tissues samples; lower RIN values were observed in adipose samples. As described by Cánovas et al. [83] (link), mRNA was purified, fragmented, and converted to cDNA. Adapters were ligated to the ends of double-stranded cDNA and PCR amplified to create libraries. These procedures were executed with the TruSeq RNA Sample Preparation kit (Illumina, San Diego, CA).
Sequencing was completed with an Illumina HiSeq analyzer that yielded 100 bp single read sequences with exception of two hypothalamus samples done earlier on an Illumina GA II sequencer that yielded 36 bp sequence reads. Sequence reads were assembled to the annotated UMD3.1 bovine reference genome (release 74; ftp://ftp.ensembl.org/pub/release-74/genbank/bos_taurus/). Quality control and RNA-Seq expression analysis was performed using procedures described by Cánovas et al. [84] (link) and using the CLC Genomics workbench software (CLC Bio, Aarhus, Denmark). Transcript levels were quantified in reads per kilobase of exon per million reads (RPKM). By normalizing for RNA length and total reads in each sample, the RPKM measure facilitated comparisons of transcript levels both within and between tissues [24] (link). In the present study, we used a threshold of RPKM ≥0.2 to select genes expressed in a given sample [22] (link), [23] (link).

Oocytes were processed as previously described [5] (link). Pig ovaries were purchased from a local slaughter house. Briefly, cumulus oocyte complexes (COCs) were aspirated from antral follicles and washed with maturation medium (Tissue Culture Medium 199) (Gibco) supplemented with 0.1% (w/v) polyvinyl alcohol (Sigma), 3.05 mM d-glucose, 0.91 mM sodium pyruvate (Sigma), 0.57 nM cysteine (Sigma), 0.5 µg/ml LH (Sigma), 0.5 µg /ml FSH (Sigma), 10 ng/ml epidermal growth factor (Sigma), 10% (v/v) porcine follicular fluid, 75 µg /ml penicillin G, and 50 µg /ml streptomycin. The COCs were then transferred to a four-well multidish (Nunc) containing 500 µl of the maturation medium covered with mineral oil and pre-equilibrated at 39°C overnight. After 42–44 h of incubation, the oocytes were released from the COCs by vigorous vortex for 4 min in TL-Hepes containing 0.1% polyvinyl alcohol and 0.1% hyaluronidase (Sigma). The polar body and associated metaphase plate of the oocytes were aspirated using glass pipettes in micromanipulation medium supplemented with 7.5 µg/ml cytochalasin B. Denuded oocytes were used as the recipients for SCNT.
The transgenic PFFs were thawed and grown to subconfluency prior to SCNT. Cells with uniform co-expression of the four fluorescent proteins were selected under fluorescent microscopy and used as donor cells that were injected into the perivitelline space of the oocytes. The oocyte-donor cell complexes were activated to fuse and become reconstructed embryos with two successive DC pulses at 1.2 kv/cm for 30 µs using an electrofusion instrument (BLS). The reconstructed embryos were cultured in embryo-development medium PZM3 at 39°C overnight and then surgically transferred to recipient gilts exhibiting natural estrus. The pregnancy status of the gilts was monitored weekly using an ultrasound scanner beginning on Day 24 after the implantation. All cloned piglets were delivered by natural birth. To monitor in vitro development, transgenic and nontransgenic SCNT embryos were cultured for 6 days until they reached the blastocyst stage.

All women underwent controlled ovarian stimulation (COS) with a GnRH antagonist fixed regimen. Bilateral antral follicles (10mm) were counted by transvaginal ultrasonography on the second day of the menstrual cycle, and women started COS treatment with gonadotrophins (Gonal-F, Merck Serono Europe Ltd or Puregon, N. V. Organon). The levels of serum progesterone (SP) were measured using an automated electrochemiluminescence immunoassay (Roche Diagnostics Elecsys Cortisol II assays and COBAS E801), and values were expressed in ng/ml. At our center, the starting dose of gonadotrophins was 150 IU/day for women aged ≤ 34 years, with BMI <24 kg/m², 6≤AFC<15, and the dosage would be increased if the woman was older (age ≥ 35 years), heavier (BMI ≥ 24 kg/m2), or had poorer ovarian reserve AFC<6 or basal FSH>10 IU/L or AMH <1 ng/ml. Conversely, if the woman is lean (BMI < 19 kg/m2) or has a good ovarian reserve (AFC≥ 15 or AMH≥ 4 ng/ml), the dosage will be reduced. Dose adjustments were determined by the physician based on individual clinical experiences. GnRH antagonists (Cetrorelix, BaxterorGanirelix, N.V.Organon) were given daily starting on day 5 or 6 of stimulation. Human chorionic gonadotrophin (Chorionic Gonadotrophin for Injection, Livzon) was injected once there were at least three follicles >17 mm in diameter or at least two follicles >18 mm in diameter. Oocyte retrieval was performed under ultrasound guidance 34-36 hours after triggering.

Conventional agonist or antagonist stimulation protocols were used for ovarian stimulation as previously described (Cai et al., 2017 (link)). The initial and ongoing dosage was determined according to patients’ age, antral follicle count (AFC), BMI, and ovarian response. An intramuscular injection of human chorionic gonadotropin (4000–6000 IU, hCG; Livzen, China) or a subcutaneous injection of recombinant human chorionic gonadotropin (250 μg, Ovidrel, Merck-Serono, Switzerland) was administrated for final triggering when at least one follicle reached a mean diameter of 18 mm. Ovum puncture under transvaginal ultrasound guidance for oocyte retrieval was performed 34–36 h after hCG injection.
Routine IVF protocol in our center was carried out (Jiang et al., 2022 (link)). Cumulus-oocyte complexes were co-cultured with approximate 1.5–3 X 10⁵ progressively motile spermatozoa in pre-equilibrated fertilization culture medium (K-SIFM, Cook) under mineral oil in traditional incubators (C200, Labotect) at 37°C, 6% CO2 and 5% O2 in a humidified atmosphere. After 4 h co-culture, oocytes were denuded and cultured individually in preequilibrated Cleavage Medium (K-SICM, Cook). The culture system and the procedure of semen preparation were kept unchanged in the period of study. Fertilization was determined according to the presence of two pronuclei (2 PN) about 17 h post insemination. It should be confirmed 2 h later if no obvious pronuclei could be observed.