Differential Transcript Profiles in Cumulus-Oocyte Complexes Originating from Pre-Ovulatory Follicles of Varied Physiological Maturity in Beef Cows

Small dominant follicle diameter at induced ovulation, but not at spontaneous ovulation, decreased pregnancy rate, fertilization rate, and day seven embryo quality in beef cows. We hypothesized that the physiological status of the follicle at GnRH-induced ovulation has a direct effect on the transcriptome of the Cumulus-Oocyte complex, thereby affecting oocyte competence and subsequent embryo development. The objective of this study was to determine if the transcriptome of oocytes and associated cumulus cells (CC) differed among small (≤11.7 mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7–14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). RNA sequencing data, from pools of four oocytes or their corresponding CC, revealed 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte pools from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. An additional 128, 98, and 80 DEG were identified among small versus large, small versus spontaneous, and large versus spontaneous follicle CC pools, respectively. The biological pathway “oxidative phosphorylation” was significantly enriched with DEG from small versus spontaneous follicle oocyte pools (FDR < 0.01); whereas the glycolytic pathway was significantly enriched with DEG from CC pools obtained from large versus small follicles (FDR < 0.01). These findings collectively suggest that altered carbohydrate metabolism within the Cumulus-Oocyte complex likely contributes to the decreased competency of oocytes from small pre-ovulatory follicles exposed to an exogenous GnRH-induced gonadotropin surge.

Assessment of Estradiol-Induced Gonadotropin Surge Generation: Preliminary Results From a Protocol of Graded Transdermal Estradiol Dosing

In normally-cycling women, high midcycle estradiol (E2) concentrations (&gt; 200-300 pg/ml) derived from the dominant follicle trigger an approximately 10-fold increase in LH and 4-fold increase in FSH levels—the gonadotropin surge—over about 48 hours. In prenatally-androgenized female rats and sheep (animal models with polycystic ovary syndrome [PCOS]-like features), high-dose estrogen fails to initiate gonadotropin surges. However, it remains unclear whether E2-induced gonadotropin surge generation is defective in PCOS. Baird et al., induced LH surges in 12 women with PCOS and 6 normally-cycling controls (studied in the early follicular phase) with 3 days of high-dose oral ethinyl E2, and the LH peak magnitude was similar in both groups (JCEM 1977;45:798-801). However, the dose of ethinyl E2 in this study (200 mcg/day) was markedly supraphysiological, and the dosing schedule did not mimic the normal, gradual increase in E2 levels observed in normal follicular phases. To further assess potential impairments in E2 augmentation of gonadotropin secretion in PCOS, we developed a study protocol involving graded transdermal E2 dosing (with dose adjustments as needed) to gradually achieve serum E2 concentrations approximating 300-400 pg/ml. Herein we present our early experience with this protocol in six normally-cycling, non-hyperandrogenic women: median (range) age 23.1 (19.8–29.0) years, body mass index 20.7 (19.2–27.2) kg/m2, calculated serum free testosterone 2.7 (2.4–5.3) pg/ml. Study subjects began transdermal E2 on cycle day 4 with the following dose-escalation protocol: 0.2 mg/d x 1 day, then 0.3 mg/d x 1 day, then 0.4 mg/d x 1 day, then 0.5 mg/d. Daily serum E2 measurements informed dose adjustments (e.g., a measured E2 level &gt; 400 pg/ml prompted a dose reduction; a measured E2 level &lt; 250 pg/ml while on the 0.5 mg/d dose prompted a dose increase to 0.6 mg/d). In these six women, daily E2 values (pg/ml) were as follows: baseline 31 (17–36) (median [IQR]) on cycle day 4 (the morning of E2 initiation); 205 (135–240) on day 5; 159 (135–270) on day 6; 263 (202–363) on day 7; 276 (239–351) on day 8; 328 (307–367) on day 9; 333 (269–402) on day 10; and 328 (253–354) on day 11. Median transdermal E2 doses (mg/d) on days 5 through 11 were 0.2, 0.3, 0.4, 0.35, 0.4, 0.4, and 0.4, respectively. Morning serum LH concentrations (median [IQR]) were 5.2 (3.5–6.1) IU/L immediately before E2 initiation (cycle day 4), decreased to a nadir of 1.5 (1.0–2.4) IU/L after 2 days of E2 (cycle day 6), thereafter increasing 8.1-fold to a peak of 12.1 (8.1–18.3) IU/L after 5 days of E2 (cycle day 9). FSH similarly changed from baseline median 3.5 (3.4–4.2) IU/L to nadir median of 1.6 (1.2–1.8) IU/L after 2 days of E2, thereafter increasing again to 3.5 (2.6–4.0) IU/L after 5 days of E2. We conclude that this experimental protocol may be useful to investigate potential defects in E2-induced LH surge generation in PCOS.

Vaginal Bleeding in an Infant with Extreme Prematurity

Background. Minipuberty of infancy refers to the transient activation of the hypothalamic-pituitary-gonadal (HPG) axis during the first few months of life. Studies have documented a more exaggerated and prolonged gonadotropin surge in preterm infants compared with term infants. We present a case of minipuberty presenting with vaginal bleeding at the corrected age of 3 months of life. Case Presentation. A former 23 + 6-week infant presented with intermittent vaginal bleeding in the diaper at the corrected age of 3 months. Physical exam showed bilateral breast buds of 0.5 cm–1 cm with no signs of pubarche. Investigations showed pubertal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol. As she was impressed to have exaggerated minipuberty due to extreme prematurity, no intervention was given. Repeated hormonal workup at the corrected age of 8 months showed decreasing trend of gonadotropin and estradiol levels. Vaginal bleeding resolved, and breast buds also regressed clinically. Conclusion. Our case illustrated that the robust surge of gonadotropin in an ex-premature infant can in fact result in endometrial maturation and present as vaginal bleeding. Though the mechanism of this alteration in the HPG axis in prematurity is not clearly understood, pediatricians should be aware of the benign and self-limiting nature of this phenomenon and avoid unnecessary intervention.

Cyclins regulating oocyte meiotic cell cycle progression†

Oocyte meiotic maturation is a vital and final process in oogenesis. Unlike somatic cells， the oocyte needs to undergo two continuous meiotic divisions (meiosis I and meiosis II) to become a haploid gamete. Notably, oocyte meiotic progression includes two rounds of unique meiotic arrest and resumption. The first arrest occurs at the G2 (germinal vesicle) stage and meiosis resumption is stimulated by a gonadotropin surge; the second arrest takes place at the metaphase II stage, the stage from which it is released when fertilization takes place. The maturation-promoting factor, which consists of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDK1), is responsible for regulating meiotic resumption and progression, while CDK1 is the unique CDK that acts as the catalytic subunit of maturation-promoting factor. Recent studies showed that except for cyclin B1, multiple cyclins interact with CDK1 to form complexes, which are involved in the regulation of meiotic progression at different stages. Here, we review and discuss the control of oocyte meiotic progression by cyclins A1, A2, B1, B2, B3, and O.

Morphological study of apoptosis in granulosa cells and ovulation in a model of atresia in rat preovulatory follicles

SummaryPrevious studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus–oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.

Effect of preovulatory follicle size on cumulus cell and follicular wall transcript abundance in beef cows

Gonadotropin releasing hormone (GnRH)-induced ovulation of small dominant follicles decreased pregnancy rates and increased late embryonic/fetal mortality in postpartum beef cows, which could be caused by inadequate oocyte competence and(or) maternal environment. Previous studies revealed that dominant follicle size at GnRHinduced ovulation in beef cows may affect oocyte competence, as higher fertilization rates and higher embryo quality were achieved as dominant follicle diameter at insemination increased. In addition, higher pregnancy rates and increased concentrations of circulating preovulatory estradiol and postovulatory progesterone were observed in recipient cows induced to ovulate large compared to small dominant follicles, which may affect the establishment of pregnancy. The objectives of the current study were to determine the effects of preovulatory follicle size and physiological status on the cumulus cell transcriptome (Experiment 1) and determine the effects of preovulatory follicle size and steroidogenic capacity (i.e. concentrations of circulating estradiol) on the dominant follicle wall transcriptome collected before the preovulatory gonadotropin surge (Experiment 2). In Experiment 1, ovulation was synchronized in suckled beef cows by administering an injection of GnRH on d-9, prostaglandin F2a (PG) on d-2, and a second injection of GnRH on d0. The dominant follicle was trans-vaginally aspirated on d1 and cumulus-oocyte complexes (COC) were collected. The cumulus cells were removed from the oocytes and assigned to one of the following follicle classifications based on follicle diameter and estrus expression: small (n=6; less than 11.7mm; no estrus expression), large (n=6; greater than 12.5 mm; no estrus expression), or spontaneous (n=5; 11.6-13.9 mm; estrus expression and endogenous gonadotropin surge). RNA was extracted from cumulus cells collected from pools of four oocytes and sequenced before being aligned to the Bos taurus genome (UMD3.1). When comparing the cumulus cell transcriptome of small versus large preovulatory follicles, 430 transcripts were more abundant at a false discovery rate (FDR) less than 0.10 in small follicles and 454 were more abundant in large follicles. The glycolytic pathway was enriched in the cumulus cells of large follicles compared to small follicles. In the small versus spontaneous preovulatory follicle comparison, 597 transcripts were more abundant in small follicles and 1012 transcripts were more abundant in spontaneous follicles. The steroid biosynthesis pathway (i.e. cholesterol synthesis) was enriched in the cumulus cells of spontaneous compared to small follicles. In the large versus spontaneous preovulatory follicle comparison, 541 transcripts were more abundant in cumulus cells from large follicles and 951 were more abundant in cumulus cells from spontaneous follicles. In summary, a greater abundance of transcripts encoding for members of the glycolytic pathway in large follicles and transcripts encoding for members of the steroid biosynthesis pathway in the cumulus cells of spontaneous compared to small follicles indicate that oocytes from small follicles may be less competent. Preovulatory follicle development was synchronized in Experiment 2 by administering GnRH on d-9 and PG on d-2 to non-lactating beef cows. The ovaries were harvested at slaughter 48 hr after PG (d0) and the dominant follicle collected. RNA was extracted from approximately half of the follicle wall and sequenced. Cows were divided into two classifications based on dominant follicle diameter at collection: small (n=4; less than 11.5 mm) and large (n=7; greater than 12.5 mm). Nine transcripts were more abundant in small follicles, and two transcripts were more abundant in large follicles. No significant pathways were found. The same 11 follicle walls were also divided into two classifications based on concentration of serum estradiol at 48 hr after PG: low (n=6; less than 4.0 pg/ml) and high (n=5; greater than or equal to 4.0 pg/ml). In the low estradiol classification, 281 transcripts were more abundant in the follicle wall and 40 were more abundant in the follicle wall of the high estradiol classification. No significant pathways were found. Transcripts were analyzed with PANTHER to find significant gene ontology (GO) terms. Differentially abundant transcripts in low estradiol follicles were more highly associated with mitosis, chromosome segregation, and regulation of biological processes. In summary, a small number of transcripts were differentially abundant in the follicle wall of small versus large dominant follicles prior to the preovulatory gonadotropin surge and no specific pathways were identified that might provide insight into how the physiological maturity of a dominant follicle can affect pregnancy rate. Comparisons made between follicle walls from follicles with low or high serum estradiol found a larger number of transcripts were more highly abundant in the low estradiol follicles, possibly indicating that a higher level of transcription is taking place in the low estradiol follicles, and therefore are less mature.