Z. Roth

Impaired reproduction in heat-stressed cattle: basic and applied aspects

Summer heat stress (HS) is a major contributing factor in low fertility in lactating dairy cows in hot environments. Although modern cooling systems are used in dairy farms, fertility remains low. This review summarizes the ways in which the functioning of various parts of the reproductive system of cows exposed to HS is impaired. The dominance of the large follicle is suppressed during HS, and the steroidogenic capacity of theca and granulosa cells is compromised. Progesterone secretion by luteal cells is lowered during summer, and in cows subjected to chronic HS, this is also reflected in lower plasma progesterone concentration. HS has been reported to lower plasma concentration of LH and to increase that of FSH; the latter was associated with a drastic reduction in plasma concentration of inhibin. HS impairs oocyte quality and embryo development, and increases embryo mortality. High temperatures compromise endometrial function and alter its secretory activity, which may lead to termination of pregnancy. In addition to the immediate effects, delayed effects of HS have been detected as well. Among them, altered follicular dynamics, suppressed production of follicular steroids, and low quality of oocytes and developed embryos. These may explain the low fertility of cattle during the cool autumn months. Hormonal treatments improve low summer fertility to some extent but not sufficiently for it to equal winter fertility. A limiting factor is the inability of the high-yielding dairy cow to maintain normothermia. A hormonal manipulation protocol, which induces timed insemination, has been found to improve pregnancy rate and to reduce the number of days open during the summer.

Involvement of apoptosis in disruption of developmental competence of bovine oocytes by heat shock during maturation.

Abstract Various pathological stimuli such as radiation, environmental toxicants, oxidative stress, and heat shock can initiate apoptosis in mammalian oocytes. Experiments were performed to examine whether apoptosis mediated by group II caspases is the cause for disruption of oocyte function by heat shock applied during maturation in cattle. Bovine cumulus-oocyte complexes (COCs) were cultured at 38.5, 40, or 41°C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were at 38.5°C and 5% (v/v) CO2 for all treatments. In the first experiment, exposure of COCs to thermal stress during the first 12 h of maturation reduced cleavage rate and the number of oocytes developing to the blastocyst stage. In the second experiment, a higher percentage of TUNEL-positive oocytes was noted at the end of maturation for oocytes matured at 40 and 41°C than for those at 38.5°C. In addition, the distribution of oocytes classified as having high (>25 intensity units), medium (15–25 intensity units), and low (<15 intensity units) caspase activity was affected by treatment, with a greater proportion of heat-shocked oocytes having medium or high activity. In the third experiment, COCs were placed in maturation medium with vehicle (0.5% [v/v] DMSO) or 200 nM z-DEVD-fmk, an inhibitor of group II caspases. The COCs were matured at 38.5 or 41°C, fertilized and cultured for 8 days. The inhibitor blocked the effect of heat shock on cleavage rate and the percentage of oocytes and cleaved embryos developing to the blastocyst stage. In conclusion, heat shock during oocyte maturation can promote an apoptotic response mediated by group II caspases, which, in turn, leads to disruption of the oocytes capacity to support early embryonic development following fertilization.

Administration of Prostaglandin F2α During the Early Bovine Luteal Phase Does Not Alter the Expression of ET-1 and of Its Type A Receptor: A Possible Cause for Corpus Luteum Refractoriness

Abstract Luteal regression is initiated by prostaglandin F2α (PGF2α). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF2α. Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF2α-induced luteolysis. Therefore, in this study, we compared the effects of PGF2α administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF2α receptors (PGF2α-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF2α analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF2α analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF2α injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450scc) was only affected when PGF2α was administered during midcycle. Nevertheless, PGF2α elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF2α-R was transiently affected. Such effects probably result from PGF2α acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF2α, possibly the endothelium, could yet be nonresponsive during the early luteal phase.

Seasonal effects on gene expression, cleavage timing, and developmental competence of bovine preimplantation embryos

We examined the association between season and expression of genes involved in early embryonic development with an emphasis on cleavage rate and timing of the first embryonic cleavage. In Exp. 1, oocytes were aspirated during the cold (Dec-Apr) and hot (May-Nov) seasons. Matured oocytes were chemically activated and cultured in vitro. The developmental peak to the two- and four-cell stages occurred earlier, with a higher proportion of first-cleaved embryos, during the cold season relative to the hot season (P<0.01). In Exp. 2, a time-lapse system was employed to characterize the delayed cleavage noted for the hot season. Cleavage to the two-cell stage occurred in two distinct waves: early cleavage occurred between 18 and 25 h post activation, and late cleavage occurred between 27 and 40 h post activation. In Exp. 3, oocytes were aspirated during the cold and hot seasons, matured in vitro, fertilized, and cultured for 8 days. In each season, early- and late-cleaved two-cell stage embryos were collected. Total RNA was isolated, and semi-quantitative and real-time PCRs were carried out with primers for GDF9, POU5F1, and GAPDH using 18S rRNA as the reference gene. In both seasons, the expression of all examined genes was higher (P<0.05) in early- versus late-cleaved embryos. POU5F1 expression was higher (P<0.05) in early-cleaved embryos developed in the cold season versus the hot season counterparts. The findings suggest a deleterious seasonal effect on oocyte developmental competence with delayed cleavage and variation in gene expression.

Sphingosine 1-Phosphate Protects Bovine Oocytes from Heat Shock During Maturation

Abstract Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that can block apoptosis by counteracting the proapoptotic effects of ceramide. Experiments were performed to evaluate whether S1P blocks the disruption in oocyte developmental competence caused by heat shock. Cumulus-oocyte complexes (COCs) were placed in maturation medium and cultured at 38.5 or 41°C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were performed at 38.5°C. Heat shock during the first 12 h of maturation reduced cleavage rate, the number of oocytes developing to the blastocyst stage, and the percentage of cleaved embryo that subsequently developed to blastocysts. Addition of 50 nM S1P to maturation medium had no effect on oocytes matured at 38.5°C but blocked effects of thermal stress on cleavage and subsequent development. The blastocysts formed at Day 8 did not differ between S1P and control groups in caspase activity, total cell number, or percentage of cells that were apoptotic. Blocking endogenous generation of S1P by addition of 50 nM N1N-dimethylsphingosine, a sphingosine kinase inhibitor, reduced or tended to reduce cleavage rate and blastocyst development regardless of whether maturation of COCs was at 38.5 or 41°C. Results demonstrate that S1P protects oocytes from a physiologically relevant heat shock and affects oocyte maturation even in the absence of heat shock. The S1P-treated oocytes that survived heat shock and became blastocysts had a normal developmental potential as determined by caspase activity, total cell number, and percentage of apoptotic cells. Thus, modulation of developmental competence of oocytes using S1P may be a useful approach for enhancing fertility in situations where developmental competence of oocytes is compromised.

Seasonal Effect on Germinal Vesicle-Stage Bovine Oocytes Is Further Expressed by Alterations in Transcript Levels in the Developing Embryos Associated with Reduced Developmental Competence

ABSTRACT Physiological perturbations of bovine follicle-enclosed oocytes during the lengthy period of follicular development can lead to reduced oocyte developmental competence. It is suggested that heat stress-induced alterations in germinal vesicle (GV)-stage oocytes are further expressed in the transcriptional levels of genes involved in oocyte maturation and early embryonic development. Bovine oocytes were collected during cold (December–April) and hot (May–November) seasons, matured, fertilized, and cultured in vitro. The percentage of fertilized oocytes cleaving to the 2- to 4-cell stage was higher in the cold vs. hot season (89.0% ± 2.63% vs. 75% ± 2.63%, respectively; P < 0.05), as was the percentage of cleaved embryos further developing to blastocysts (26.6% ± 0.9% vs. 10.1% ± 1.8%, respectively; P < 0.05). Total RNA and poly(A) mRNA of oocytes and developing embryos were isolated and subjected to semiquantitative and real-time PCR for MOS, GDF9, and POU5F1 genes. In GV-stage oocytes, their mRNA levels did not differ between the seasons. However, following maturation, mRNA levels were higher in oocytes collected in the cold season (P < 0.05). In 4-cell-stage embryos, GDF9 and POU5F1 showed opposite mRNA patterns between seasons (higher and lower levels, respectively) in the hot season (P < 0.05). In both 8-cell-stage embryos and blastocysts, POU5F1 expression was lower during the hot season (P < 0.05). Exposing the ovarian pool of oocytes to environmental stress appears to impair maternal mRNA storage and/or the mechanism of transcription renewal, in turn affecting embryo gene expression before and after embryonic genome activation. Such impairment might partially explain the carry-over effect of summer heat stress on dairy cow conception rates.

Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush

BACKGROUND Vitrification is becoming the method of choice for embryo cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of liquid nitrogen (LN) contamination. METHODS An innovative vitrification method that combines LN slush and sealed pulled straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with LN. RESULTS Ultrarapid cooling of murine embryos at 32 200 degrees C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 +/- 3.5%, 106/196) than cooling at 1700 degrees C/min in 0.25 ml straws (10 +/- 2.1%, 21/197) (P < 0.05). Embryos at the 2-cell stage cryopreserved in 75% vitrification solution (VS) (100% VS contains approximately 5 M ethylene glycol, 0.6 M trehalose and 6% w/v bovine serum albumin) in SPS formed blastocysts at a higher rate (79 +/- 3.6%, 99/126) than cryopreservation in 100% VS (31 +/- 6.5%, 16/51), however, this was not significantly different from the fresh control group (88 +/- 4.6%, 43/49). Early stage embryos at the 2 pronuclei- and 4-8-cell stage formed blastocysts at rates of 68 +/- 4.5 and 60 +/- 3.7%, respectively, after vitrification in 87.5% VS. CONCLUSIONS This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

The antioxidant epigallocatechin gallate (EGCG) moderates the deleterious effects of maternal hyperthermia on follicle-enclosed oocytes in mice

Hyperthermia-induced oxidative stress is one of the mechanisms suggested to underlie loss of developmental competence in mouse embryos. In this study, we examined whether pretreatment with the antioxidant epigallocatechin gallate (EGCG) can alleviate the negative effects of hyperthermia on developmental competence of the ovarian pool of oocytes and improve embryonic development. Female mice (CB6F1) were synchronized (eCG+hCG) and injected with 0.4 ml EGCG (100 mg/kg body weight) or with saline. Both EGCG- and saline-treated mice were exposed to heat stress (HS; 40 degrees C, 65% RH) or kept under normothermal conditions (Control; 22 degrees C, 45% RH). In vivo-derived zygotes were recovered 20 h after hCG administration and cultured in vitro. Maternal hyperthermia attenuated embryonic cleavage rate in association with further disruption in embryonic early cleavage and subsequently, with embryonic development. While pretreatment with EGCG did not affect the proportion of zygotes that cleaved to the two-cell stage, it appeared to moderate the effect of hyperthermia on both cleavage timing and developmental rate, as reflected by an increased rate of early cleaved embryos and blastocyst formation. Blastocyst developmental competence was also improved, as indicated by the increased total cell number and percentage of embryos that underwent hatching, in association with reduced apoptotic status, as reflected by the percentage of TUNEL-positive cells and intensity of caspase activity for the HS-EGCG embryos vs. HS-saline ones. In summary, while hyperthermia disrupts the competence of the follicle-enclosed oocyte, in vivo administration of the antioxidant EGCG improves developmental competence and the quality of the embryos that develop from these oocytes.

Ovarian cysts in high-yielding dairy cows.

We examined the hormonal and morphologic changes associated with ovarian cyst formation in high-yielding dairy cows. Follicle fluid was aspirated from 90 cysts and 15 preovulatory and 18 subordinate follicles and used for hormonal determination. Pieces of cystic wall were subjected to morphologic and immunohistochemical evaluation. Cysts were characterized by low concentrations of insulin, insulin-like growth factor-I (IGF-I), and glucose and high activity of IGF binding proteins (IGFBPs). Insulin and IGF-I levels were (mean+/-SEM) 205+/-22 pg/mL and 146+/-42 ng/mL in preovulatory follicles and 3+/-1 pg/mL and 61+/-6 ng/mL in cysts, respectively (P<0.001). Insulin-like growth factor-binding proteins activity was about 10 times higher in cysts than in preovulatory follicles. Cysts were classified into three types according to their estradiol-to-progesterone (E/P) ratio. Type 1 cysts (n=23) exhibited the highest E/P ratio (10.8+/-2.3), partial loss of granulosa cells, and severe morphologic changes in the theca interna. Expression of P(450) side-chain cleavage and P(450) 17 alpha-hydroxylase was noted in theca cells and expression of inhibin-alpha in granulosa cells. Type 2 cysts (n=35) had a low E/P ratio (0.07+/-0.02), and patches of luteal-like tissue in the cystic wall. Type 3 cysts (n=32) had an E/P ratio of 0.91+/-0.17, and no recognizable granulosa or theca cells. In summary, intrafollicular steroid levels as expressed by E/P ratio, together with IGF-I and insulin levels and morphologic changes in the follicular wall, may serve as accurate cyst-classification parameters. Because IGF-I and/or insulin play an essential role in the final stage of follicle development, it can be speculated that abnormal levels of these metabolic hormones might lead to follicle dysfunction, resulting in follicular regression or cyst formation.

Incorporation of Coenzyme Q10 into Bovine Oocytes Improves Mitochondrial Features and Alleviates the Effects of Summer Thermal Stress on Developmental Competence

ABSTRACT Environmental stress-induced alterations in oocyte mitochondria are suggested to deleteriously affect developmental competence of the ovarian pool of oocytes. We examined the association between seasonal effects on oocyte developmental competence and mitochondrial distribution, polarization, mitochondrial DNA (mtDNA) content, and RNA expression, and whether the incorporation of coenzyme Q10 (CoQ10) might improve these effects. Bovine oocytes were collected during the summer (June–August), fall (September–November), and winter (December–May), matured in vitro with or without 50 μM CoQ10, fertilized, and cultured for 8 days. The proportion of developed blastocysts was highest in the winter, intermediate in the fall, and lowest in the summer. Matured oocytes were classified into categories I–IV according to their mitochondrial distribution pattern (MitoTracker green). The proportion of high- and low-polarized mitochondria (JC-1 assay) differed between oocyte categories but was not affected by season. On the other hand, oocyte distribution into categories differed between seasons and was affected by CoQ10, with an increased proportion of category I oocytes in the fall. Oocyte mtDNA did not differ between seasons, but expression of mitochondrion-associated genes involved in the respiratory chain (ND2, SDHD, CYTB, COXII, ATP5B, and TFAM) did. Coenzyme Q10 increased the expression of CYTB, COXII, and ATP5B and the proportions of blastocysts developed in the fall. In summary, season-induced alterations in mitochondrial functions might explain, in part, the reduced oocyte developmental competence. It seems that in the fall, under modest harm, CoQ10 incorporation can alleviate these deleterious effects somewhat.