Temporal dynamics of oocyte development, plasma sex steroids and somatic energy reserves during seasonal ovarian maturation in captive Murray cod Maccullochella peelii peelii

The temporal dynamics of oocyte growth, plasma sex steroids and somatic energy stores were examined during a 12 month ovarian maturation cycle in captive Murray cod Maccullochella peelii peelii under simulated natural photothermal conditions. Ovarian function was found to be relatively uninhibited in captivity, with the exception that post-vitellogenic follicles failed to undergo final maturation, resulting in widespread pre-ovulatory atresia. Seasonal patterns of oocyte growth were characterised by cortical alveoli accumulation in March, deposition of lipids in April, and vitellogenesis between May and September. Two distinct batches of vitellogenic oocytes were found in Murray cod ovaries, indicating a capacity for multiple spawns. Plasma profiles of 17β-oestradiol and testosterone were both highly variable during the maturation period suggesting that multiple roles exist for these steroids during different stages of oocyte growth. Condition factor, liver size and visceral fat stores were all found to increase prior to, or during the peak phase of vitellogenic growth. Murray cod appear to strategically utilise episodes of high feeding activity to accrue energy reserves early in the reproductive cycle prior to its deployment during periods of rapid ovarian growth.     

The effects of season and moderate nutritional restriction on ovarian function and oocyte nuclear maturation in cycling gilts

The fertility of female pigs is impaired during summer and in response to restriction of feed intake, resulting in reduced productivity of the breeding herd. This study determined the effect of season and moderate nutritional restriction on ovarian function and oocyte developmental competence of cycling gilts. Eighty prepubescent gilts were used across two seasons—summer (S: January to March) and winter (W: June to August)—and received either a high (2.5× maintenance) or a moderately restricted (1.5× maintenance) feeding level for the first 19 days of their second estrous cycle. On Day 19, ovaries were collected post-slaughter. Diameters of all surface follicles over 1 mm were measured. All follicles ≥4 mm were aspirated and cumulus–oocyte complexes underwent in vitro maturation for ∼44 hours to assess oocyte developmental competence on the basis of metaphase II (MII) attainment. Moderate dietary nutrition reduced daily liveweight gain but did not affect the ovarian follicle population or oocyte developmental competence. The number of large follicles (≥6 mm) was lower during summer (S: 10.7 ± 1.74 vs. W: 15.5 ± 1.15, P < 0.05), as was the proportion of oocytes at the germinal vesicle stage of meiosis (S: 0.06 ± 0.02 vs. W: 0.08 ± 0.02, P < 0.05). However, the proportion of oocytes attaining MII was similar in summer and winter (S: 0.72 ± 0.04 and W: 0.69 ± 0.06, P > 0.05). Intrafollicular concentrations of luteinizing hormone were higher in summer (S: 43.05 ± 6.44 vs. W: 12.05 ± 5.12 ng/mL, P < 0.001), whereas estradiol was lower (S: 1.27 ± 0.36 vs. W: 27.52 ± 5.59 ng/mL, P < 0.001). In conclusion, our data demonstrated that in summer, follicle growth beyond 6 mm is impaired during the periovulatory period, without affecting oocyte meiotic competence. Importantly, these data also demonstrated that ovarian follicle growth and the capacity of oocytes to reach MII in vitro appear unaffected by moderate nutritional restriction during the preceding estrous cycle.     

Expression of maturation genes and their receptors during in vitro maturation of sheep COCs in the presence and absence of somatic cells of cumulus origin

The purpose of this study was to investigate the effects of somatic cells of cumulus origin (sCC) on gene expression and maturation of cumulus oocyte complexes (COCs) in vitro. Good quality (i.e., healthy-looking) isolated sheep COCs were randomly divided into two treatment groups: control (COC with no sCC) and coculture (COC with sCC). Nuclear maturation statuses of oocytes were assessed after 27 hours of in vitro culture. Moreover, the expression levels of growth differentiation factor 9 (GDF9), bone morphogenetic protein (BMP)15, BMP6, bone morphogenetic protein receptor II (BMPRII), activin like kinase 5 (ALK5) (transforming growth factor β receptor 1: TGFβR1), ALK6 (BMPR1b), activin A receptor, type IIB (ActRIIB), and ALK3 (BMPR1a), as well as hyaluronan synthase 2 (HAS2) and prostaglandin endoperoxide synthase 2 (Ptgs2) in the COCs were assessed in both treatment groups after 3 h and 27 h of culture. The results showed that the proportion of metaphase II (MII) stage oocytes was significantly higher in the coculture group compared with the controls (77.21% ± 1.17 vs. 67.49% ± 1.80; P < 0.05). The relative expressions of BMPRII, ALK6, and ActRIIB in control group and GDF9 and ActRIIB in coculture group showed significant differences during culture as assessed by real time polymerase chain reaction (P < 0.05). The mean expression levels of BMPRII, ALK5, ALK6, and ActRIIB mRNA were decreased in the coculture group compared with those in the control group after 27 h of culture (P < 0.05). In conclusion, we propose that in vitro maturation of sheep COCs alone disrupted the normal gene expression levels of both TGFβ ligands and receptors, and also reduced the maturation rate. Coculture with sCC enhanced the maturation rate of oocytes concomitantly with reduced gene expression levels of a number of TGFβ ligands and receptors.     

Effects of in vitro culture of mouse fetal gonads on subsequent ovarian development in vivo and oocyte maturation in vitro

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 µm in diameter and had competence to resume meiosis in vitro . When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences ( P  < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.     

Regulation of oocyte mitochondrial DNA copy number by follicular fluid, EGF, and neuregulin 1 during in vitro maturation affects embryo development in pigs

Little is known about mitochondrial DNA (mtDNA) replication during oocyte maturation and its regulation by extracellular factors. The present study determined the effects of supplementation of maturation medium with porcine follicular fluid (pFF; 0, 10%, 20%, and 30%) on mtDNA copy number and oocyte maturation in experiment 1; the effects on epidermal growth factor (EGF; 10 ng/mL), neuregulin 1 (NRG1; 20 ng/mL), and NRG1 + insulin-like growth factor 1 (IGF1; 100 ng/mL + NRG1 20 ng/mL), on mtDNA copy number, oocyte maturation, and embryo development after parthenogenic activation in experiment 2; and effects on embryo development after in vitro fertilization in experiment 3. Overall, mtDNA copy number increased from germinal vesicle (GV) to metaphase II (MII) stage oocytes after in vitro maturation (GV: 167 634.6 ± 20 740.4 vs. MII: 275 131.9 ± 9 758.4 in experiment 1; P < 0.05; GV: 185 004.7 ± 20 089.3 vs. MII: 239 392.8 ± 10 345.3 in experiment 2; P < 0.05; Least Squares Means ± SEM). Supplementation of IVM medium with pFF inhibited mtDNA replication (266 789.9 ± 11 790.4 vs. 318 510.1 ± 20 377.4; P < 0.05) and oocyte meiotic maturation (67.3 ± 0.7% vs. 73.2 ± 1.2%, for the pFF supplemented and zero pFF control, respectively; P < 0.01). Compared with the control, addition of growth factors enhanced oocyte maturation. Furthermore, supplementation of NRG1 stimulated mitochondrial replication, increased mtDNA copies in MII oocytes than in GV oocytes, and increased percentage of blastocysts in both parthenogenetic and in vitro fertilized embryos. In this study, mitochondrial biogenesis in oocytes was stimulated during in vitro maturation. Oocyte mtDNA copy number was associated with developmental competence. Supplementation of maturation medium with NRG1 increased mtDNA copy number, and thus provides a means to improve oocyte quality and developmental competence in pigs.     

Influence of different reproductive rhythms on serum estradiol and testosterone levels,features of follicular population and atresia rate,and oocyte maturation in controlled suckling rabbits

The aim of the current work was to analyze the features of ovarian follicular population and their quality in New Zealand white rabbit does synchronized by 24-h controlled doe-litter separation before artificial insemination (AI) during all their reproductive cycles. Synchronized animals were allocated systematically in two groups. A total of 73 rabbit does (group A) were submitted to a 35-day intensive rhythm (AI on day 4 post-partum [pp] and weaning at 25 days of lactation), and 108 rabbit does (group B) were submitted to a 42-day semi-intensive rhythm (AI on day 11 pp and weaning at 35 days of lactation) during 9 months. At the mid-end of their reproductive life, a total of 26 does (5.4 parturitions), under intensive (n = 15) or semi-intensive rhythm (n = 11) were either treated in each group with 25 IU eCG 48 h before laparotomy to recover their ovaries (n = 7 for group A and n = 6 for group B) (according to the Bioethics Committee of the University) or not synchronized with the hormonal treatment (n = 8 for group A and n = 5 for group B). Blood samples were collected at the moment of ovary recovery; morphometrical parameters, number of total follicles and number of follicles ≥1 mm in size in the ovarian surface were recorded. Oocytes from follicles of one ovary were recovered and matured in TCM 199 supplemented with 10 ng/ml EGF, 100 ng/ml IGF-I and 10% FCS. The counterpart ovaries were fixed in paraformaldehyde solution for histological studies. Detection of cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) technique. Reproductive performance was affected by the rhythm used, with lower reproductive parameters in the intensive group. The average ovary height and width, the mean number of ≥1 mm follicles and the number of total follicles were similar between groups. Serum concentrations of estradiol (E₂) and testosterone (T) were significantly lower in group A vs. B (E₂: 232.4 ± 56.1 vs. 399.7 ± 53.0 pg/ml; P < 0.05 and T: 1.07 ± 0.10 vs. 1.68 ± 0.23 ng/ml; P < 0.05). No significant differences were found in follicular population or in the mean follicular apoptosis index between groups. Metaphase II rate was significantly lower in group A vs. B (48.5 ± 3.3 vs. 67.6 ± 3.7%; P < 0.01), as well as the migration rate of cortical granules (12.7 ± 2.7 vs. 38.2 ± 6.6%; P < 0.001). On the other hand, neither follicular population, nuclear maturation rate nor apoptosis rate were affected by the eCG treatment, but cytoplasmic maturation was higher in animals treated with eCG in group A (29.2% vs. 5.5%; P < 0.01). In conclusion, rabbit does under transient litter separation during their reproductive life have both their serum estradiol and testosterone concentrations and oocyte quality influenced by the intensive rhythm, leading to a decrease in reproductive parameters. Also, both intensive and semi-intensive rhythms seem to be less receptive to eCG treatment than expected.     

Repeatability of 2-wave and 3-wave patterns of ovarian follicular development during the bovine estrous cycle

Characteristics predictive of a 2-wave versus 3-wave pattern of ovarian follicular development during the interovulatory interval (IOI) were examined by ultrasonographic monitoring of 91 IOIs from 31 beef heifers. Repeatability of the wave pattern within individuals and the effects of season and age were determined using a subset of 75 IOIs from 15 heifers examined for multiple IOIs. The 2-wave pattern was detected in 62 of 91 (68%) IOIs, and the 3-wave pattern was detected in 29 of 91 (32%) IOIs. The preponderance of the 2-wave versus 3-wave pattern (P < 0.05) was not influenced by season (P = 0.61) but was even greater in the more mature age group (P = 0.02). The majority of IOIs ≤21 d was of the 2-wave pattern (88%; P < 0.05), whereas the majority of IOIs ≥22 d was of the 3-wave pattern (78%; P < 0.05). The proportion of nonalternating patterns (repeatability) was more than twofold greater than the proportion of alternating patterns (70% vs. 30%; P < 0.01). This relationship was consistent among seasons (P < 0.01) and even more marked in the more mature age group (P = 0.01). Emergence and follicular dominance of Wave 2 were delayed (P < 0.01), and the onset of corpus luteum regression was earlier (P < 0.01) in 2-wave versus 3-wave IOI. In conclusion, the duration of the IOI was predictive of the wave pattern, and the pattern was repeatable within individuals. Factors influencing the period of follicular dominance of Wave 1 in 2-wave versus 3-wave IOI may be responsible for regulating the wave pattern and may be associated with heifer maturity or relative nutritional demand during the postpubertal period. The impact of greater follicular attrition recorded in 3-wave versus 2-wave IOI on ovarian depletion and reproductive senescence is worthy of critical evaluation.     

Changes in endoplasmic reticulum structure during mouse oocyte maturation are controlled by the cytoskeleton and cytoplasmic dynein

Oocyte maturation in mouse is associated with a dramatic reorganisation of the endoplasmic reticulum (ER) from a network of cytoplasmic accumulations in the germinal vesicle-stage oocyte (GV) to a network of distinctive cortical clusters in the metaphase II egg (MII). Multiple lines of evidence suggest that this redistribution of the ER is important to prepare the oocyte for the generation of repetitive Ca2+ transients which trigger egg activation at fertilisation. The aim of the current study was therefore to investigate the timecourse and mechanism of ER reorganisation during oocyte maturation. The ER is first restructured at the time of GV-breakdown (GVBD) into a dense network of membranes which envelop and invade the developing meiotic spindle. GVBD is essential for the initiation of ER reorganisation, since ER structure does not change in GV-arrested oocytes. ER reorganisation is also prevented by the microtubule inhibitor nocodazole and by the inhibition of cytoplasmic dynein, a microtubule-associated motor protein. ER redistribution at GVBD is therefore dynein-driven and cell cycle-dependent. Following GVBD the dense network of ER surrounds the spindle during its migration to the oocyte cortex. Cortical clusters of ER are formed close to the time of, but independently of the metaphase I-metaphase II transition. Formation of the characteristic ER clusters is prevented by the depolymerisation of microfilaments, but not of microtubules. These experiments reveal that ER reorganisation during oocyte maturation is a complex multi-step process involving distinct microtubule- and microfilament-dependent phases and indicate a role for dynein in the cytoplasmic changes which prepare the oocyte for fertilisation.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Analytical and experimental studies on the relationship between Na+, K+, and water uptake during volume increases associated with Fundulus oocyte maturation in vitro

Summary Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17-hydroxy-20-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors-Na⁺ and K⁺-also rose, the increase in K⁺ being twice that of Na⁺. The influx of K⁺ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K⁺, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K⁺ in a concentration-dependent manner. The mechanism by which K⁺ accumulates in the follicle was insensitive to ouabain, so that a typical Na⁺, K⁺-ATPase mechanism does not appear to be involved. The ability of external K⁺ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation. Removal of all associated somatic cells prior to maturation prevented subsequent steroid-initiated hydration but not maturation. The results suggest that K⁺ may be translocated from surrounding granulosa cells to the oocyte via gap junctions during maturation.Abbreviations GVBD germinal vesicle breakdown     

Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development

Adiponectin is one of the most important, recently discovered adipocytokines that acts at various levels to control male and female fertility through central effects on the hypothalamus-pituitary axis or through peripheral effects on the ovary, uterus, and embryo. We studied simultaneous changes in the gene expression pattern of adiponectin and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) in granulosa and theca cells, cumulus-oocyte complex, and in corpus luteum in healthy bovine (Bos tarus) follicles at different stages of development. The expression levels of adiponectin, AdipoR1, and AdipoR2 mRNA were lower (P < 0.05) in granulosa and cumulus cells in comparison with that in theca cells and oocyte. In contrast with the oocyte, AdipoR1 in granulosa, theca, and luteal cells was expressed (P < 0.05) more than AdipoR2. Adiponectin expression increased (P < 0.05) in granulosa cells and in cumulus-oocyte complex during follicular development from small to large follicles. Opposite results were observed in theca cells. Expression of adiponectin was highest in the late stages of corpus luteum (CL) regression, whereas lower expression was recorded in active CL (P < 0.05). AdipoR1 and AdipoR2 expression increased during the terminal follicular growth in granulosa and theca cells (P < 0.05) and during the luteal phase progress in CL. There was positive correlation between adiponectin mRNA level in granulosa cells from large follicles and follicular fluid estradiol concentration (r = 0.48, P < 0.05) and negative correlation between adiponectin mRNA abundance in theca cells and follicular fluid progesterone concentration (r = -0.44, P < 0.05). In conclusion, we found that the physiologic status of the ovary has significant effects on the natural expression patterns of adiponectin and its receptors in follicular and luteal cells of bovine ovary.