271 EFFECT OF MARE AGE ON OOCYTE MORPHOLOGY AND DEVELOPMENTAL COMPETENCE AFTER INTRACYTOPLASMIC SPERM INJECTION

Reduced fertility in aged mares is associated with delayed early embryo development and lower pregnancy rates, potentially related to oocyte developmental competence. Human oocyte morphology has been associated with developmental potential, although comparative evidence is lacking in the mare. Exogenous FSH may be beneficial in obtaining more oocytes; however, effects on oocyte morphology and competence are unknown. Objectives were to determine if zona pellucida thickness (ZPT), ooplasm volume (OV), and perivitelline space volume (PVSV) were related to mare age or FSH treatment and to cleavage, blastocyst, and pregnancy rates after intracytoplasmic sperm injection (ICSI). Cycles with and without eFSH treatment were alternated; eFSH treatments began in diestrus with a cohort of follicles ≥20 mm. Oocytes were collected by transvaginal aspiration from follicles >30 mm from young (4 to 9 years) and old (>20 years) mares at 20 to 24 h after administration of recombinant eLH. Oocytes were cultured for 18 h in TCM-199 at 38.5�C in 6% CO2 in air. Sperm were injected 40 � 1 h after eLH, using frozen sperm from a single ejaculate. Presumptive zygotes were incubated in Dulbecco's modified Eagle's medium/F12 + 10% fetal calf serum at 38.5�C in 5% CO2, 5%O2, and 90% N2. Cleavage (≥2 cells) was recorded 48 h after ICSI. Blastocysts considered viable (formation before 9 d and good quality) were transferred nonsurgically into recipients 3 to 7 days after ovulation. Only pregnancies of fetuses with heart beats were included. Morphological parameters of oocytes (old, n = 40; young, n = 37) were obtained from photographic images taken at ICSI and analyzed by computer-assisted measurement using digital calipers (Spot Software, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Zona pellucida thickness was averaged from 2 measurements 90� to 180� apart. Ooplasm volume was calculated (4/3πr3) from the average of 2 diameters of the ooplasm 90� apart; and PVSV was calculated as the difference of the vitelline membrane volume and that of the volume at the inner volume of the ZP calculated as an oblate spheroid (4/3πa2b) from the average of 2 diameters. Zona pellucida thickness, OV, and PVSV were analyzed using 2-way ANOVA for main effects of age and treatment and 3-way ANOVA by adding cleavage as a factor. Zona pellucida thickness was less (P = 0.007) for old compared with young (least squares mean SEM of 11.4 � 0.2 and 12.3 � 0.2 µm, respectively) with no effect on cleavage, blastocyst, or pregnancy rates. Ooplasm volume was not different (P = 0.14) between old and young (309 036 � 5373 and 320 544 � 5639 µm3, respectively) and did not affect cleavage, blastocyst, or pregnancy rates. The PVSV was greater (P = 0.001) in old compared with young (157 505 � 10 853 and 102 161 � 11 388 µm3, respectively) and may be related to the lower cleavage (P = 0.03), blastocyst (P = 0.02), and pregnancy (P = 0.05) rates. Treatment with FSH had no effect (P > 0.1) on morphology or embryo development. In this study, ZPT and PVSV differed with mare age and could be of predictive value for oocyte developmental competence.

Download Full-text

Effects of age and equine follicle-stimulating hormone (eFSH) on collection and viability of equine oocytes assessed by morphology and developmental competency after intracytoplasmic sperm injection (ICSI)

Reproduction Fertility and Development ◽

10.1071/rd08210 ◽

2009 ◽

Vol 21 (4) ◽

pp. 615 ◽

Cited By ~ 32

Author(s):

J. L. Altermatt ◽

T. K. Suh ◽

J. E. Stokes ◽

E. M. Carnevale

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Follicle Stimulating Hormone ◽

Developmental Competence ◽

Perivitelline Space ◽

Central Cavity ◽

Developmental Potential ◽

Oocyte Morphology ◽

Preovulatory Follicles ◽

Morphology Parameters ◽

Stimulating Hormone

Young (4 to 9 yr) and old (≥20 yr) mares were treated with equine follicle-stimulating hormone (eFSH), and oocytes were collected for intracytoplasmic sperm injections (ICSI). Objectives were to compare: (1) number, morphology and developmental potential of oocytes collected from young v. old mares from cycles with or without exogenous eFSH and (2) oocyte morphology parameters with developmental competence. Oocytes were collected from preovulatory follicles 20 to 24 h after administration of recombinant equine LH and imaged before ICSI for morphological measurements. After ICSI, embryo development was assessed, and late morulae or blastocysts were transferred into recipients’ uteri. Cycles with eFSH treatment resulted in more follicles (1.8 v. 1.2) and more recovered oocytes (1.1 v. 0.8) than those without eFSH. Age and eFSH treatment did not effect cleavage, blastocyst and pregnancy rates. Treatment with eFSH had no effect on oocyte morphology, but age-associated changes were observed. In old mares, zona pellucidae (ZP) were thinner than in young mares, and perivitelline space and inner ZP volume (central cavity within the ZP) were larger and associated with oocytes that failed to develop. These results suggest that administration of eFSH can increase the number of oocytes collected per cycle. Oocyte morphology differed with age and was associated with developmental competence.

Download Full-text

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

Development ◽

10.1242/dev.128.7.1119 ◽

2001 ◽

Vol 128 (7) ◽

pp. 1119-1126 ◽

Cited By ~ 6

Author(s):

T.L. Rankin ◽

M. O'Brien ◽

E. Lee ◽

K. Wigglesworth ◽

J. Eppig ◽

...

Keyword(s):

Zona Pellucida ◽

Embryonic Stem ◽

Single Copy ◽

Targeted Mutagenesis ◽

Developmental Competence ◽

Normal Male ◽

Null Mouse ◽

Developmental Potential ◽

Early Embryos

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.

Download Full-text

Mutations in ZP4 are associated with abnormal zona pellucida and female infertility

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-207170 ◽

2021 ◽

pp. jclinpath-2020-207170

Author(s):

Xiaoli Wei ◽

Youzhu Li ◽

Qicai Liu ◽

Wensheng Liu ◽

Xiaohong Yan ◽

...

Keyword(s):

Embryo Development ◽

Zona Pellucida ◽

Intracytoplasmic Sperm Injection ◽

Treatment Strategies ◽

Suction Pressure ◽

Human Female ◽

Female Reproduction ◽

Efficient Treatment ◽

Whole Exome

BackgroundThe zona pellucida (ZP) of human oocytes plays essential protective roles in sperm–egg interactions during fertilisation and embryo development. ZP4-null female rabbits exhibit a thin and irregular ZP, which severely impairs embryo development and fertility. However, the effects of ZP4 defect on human female reproduction remain unknown.Methods and resultsWe performed whole-exome sequencing in 26 female patients with abnormal (thin and irregular) ZP and identified heterozygous variants in ZP4 (OMIM: 613514) from 3 patients (approximately 11%). No ZP4 variant was found in the 30 control women with proven fertility. We constructed ZP4-mutated plasmids and found that the variants reduced the secretion of ZP4 in vitro. Lower suction pressure facilitated egg retrieval, and intracytoplasmic sperm injection (ICSI) was a desirable treatment for ZP4-mutated patients with abnormal ZP.ConclusionsWe identified ZP4 as a novel gene for human abnormal ZP and found that lower suction pressure and ICSI are efficient treatment strategies.

Download Full-text

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab161 ◽

2016 ◽

Vol 28 (2) ◽

pp. 210

Author(s):

P. Hugon ◽

J. Lamy ◽

E. Corbin ◽

P. Mermillod ◽

M. Saint-Dizier

Keyword(s):

Corpus Luteum ◽

Embryo Development ◽

Zona Pellucida ◽

Developmental Stages ◽

Early Embryo ◽

Developmental Competence ◽

Control Group ◽

Vitro Fertilization ◽

Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

Download Full-text

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab35 ◽

2011 ◽

Vol 23 (1) ◽

pp. 124

Author(s):

C. Feltrin ◽

M. Machado ◽

L. M. V. Queiroz ◽

M. A. S. Peixer ◽

P. F. Malard ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Zona Pellucida ◽

Blastocyst Stage ◽

Aggregation State ◽

Developmental Potential ◽

In Vitro Development ◽

Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

Download Full-text

Genetic merit for fertility traits in Holstein cows: VI. Oocyte developmental competence and embryo development

Journal of Dairy Science ◽

10.3168/jds.2018-15813 ◽

2019 ◽

Vol 102 (5) ◽

pp. 4651-4661 ◽

Cited By ~ 2

Author(s):

S.G. Moore ◽

S.B. Cummins ◽

S. Mamo ◽

P. Lonergan ◽

T. Fair ◽

...

Keyword(s):

Embryo Development ◽

Developmental Competence ◽

Holstein Cows ◽

Genetic Merit ◽

Oocyte Developmental Competence ◽

Fertility Traits

Download Full-text

GLP is critical for oogenesis exhibiting a G9A-independent role in transcriptional repression

10.1101/2021.12.20.473460 ◽

2021 ◽

Author(s):

Hannah Demond ◽

Courtney W Hanna ◽

Juan Castillo-Fernandez ◽

Fatima Santos ◽

Evangelina K Papachristou ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Transcriptional Repression ◽

Knockout Mouse ◽

Conditional Knockout ◽

Developmental Competence ◽

Oocyte Developmental Competence ◽

Gene Dysregulation ◽

Conditional Knockout Mouse

GLP (EHMT1) functions as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with G9A (EHMT2). Here, we investigated the role of GLP in oocyte and embryo development in comparison to G9A using oocyte-specific conditional knockout mouse models (G9a cKO, Glp cKO, G9a-Glp cDKO). Loss of GLP in oogenesis severely impairs oocyte maturation, fertilization and embryo development, resulting in lethality before embryonic day E12.5. In contrast, loss of G9A has a milder effect with a proportion of embryos producing viable offspring. The Glp cKO also showed loss of G9A protein and, hence, was phenotypically very similar to the G9a-Glp cDKO. H3K9me2 was equally depleted in all cKO genotypes, whereas H3K9me1 was decreased only in Glp cKO and G9a-Glp cDKO oocytes. Furthermore, the transcriptome, DNA methylome and proteome were markedly more affected in G9a-Glp cDKO than G9a cKO oocytes, demonstrating that in the absence of GLP there are widespread epigenetic and gene expression changes in the oocyte independent of H3K9me2. Gene dysregulation with coupled changes in DNA methylation suggest localised loss of chromatin repression, resulting in upregulated protein expression. Together, our findings demonstrate that GLP can function independently of G9A in the oocyte and is required for oocyte developmental competence.

Download Full-text

313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab313 ◽

2015 ◽

Vol 27 (1) ◽

pp. 245 ◽

Cited By ~ 1

Author(s):

N. W. Santiquet ◽

A. F. Greene ◽

W. B. Schoolcraft ◽

R. L. Krisher

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Subsequent Development ◽

Developmental Competence ◽

Nuclear Maturation ◽

Oocyte Developmental Competence ◽

Oocyte Collection ◽

Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

Download Full-text

Oocyte developmental competence in a bovine model of reproductive aging

Reproduction ◽

10.1530/rep-07-0021 ◽

2007 ◽

Vol 134 (2) ◽

pp. 233-239 ◽

Cited By ~ 49

Author(s):

Pritpal S Malhi ◽

Gregg P Adams ◽

Reuben J Mapletoft ◽

Jaswant Singh

Keyword(s):

Embryo Transfer ◽

Age Groups ◽

Blastocyst Stage ◽

Pregnancy Rates ◽

Developmental Competence ◽

Corpora Lutea ◽

Reproductive Aging ◽

Embryo Production ◽

Embryo Survival ◽

Oocyte Developmental Competence

The study was designed to test the hypothesis that aging in cattle is associated with reduced developmental competence of oocytes. The hypothesis was tested by comparing embryo production and pregnancy rates between 13- to 16-year-old cows (n = 6 in Year 1 and n = 9 in Year 2) and their 3- to 6-year-old young daughters (n = 8 in Year 1 and n = 9 in Year 2) after superovulation and transfer of embryos into an unrelated group of young recipients. Embryos were transferred into 2- to 5-year-old recipient cows (n = 99) as singletons (n = 45) or in pairs (n = 54 pairs). Embryo survival in recipients was determined by ultrasonography and by the number of calves born. Between old versus young cows, the number of ovulations (31 ± 4 vs 38 ± 3; P = 0.2) and the number of corpora lutea (25 ± 3 vs 29 ± 2; P = 0.3) did not differ, but fewer (P = 0.04) embryos were recovered from old cows (6 ± 2) than their daughters (12 ± 2). A higher proportion (P < 0.0001) of unfertilized oocytes/uncleaved zygotes were recovered from old cows (222/312, 71%) than their daughters (119/316, 38%). Among the embryos recovered, the proportion of International Embryo Transfer Society Grades 1–2 embryos was similar (P = 0.9) between old (59/90, 66%) and young cows (130/194, 67%). The survival of embryos after transfer into recipients, and the proportion of calves born were also similar between old and young cows. In conclusion, recovery of fewer embryos and a greater proportion of unfertilized oocytes/uncleaved zygotes suggest reduced developmental competence of oocytes from old cows, but there was no difference between age groups in embryo survival after the morula/blastocyst stage.

Download Full-text