A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1.

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability.

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.

Pregnancy rates and metabolic profiles in cattle treated with propylene glycol prior to embryo transfer.

The objective of this study was to determine the effect of a sustained propylene glycol administration to recipients of frozen/thawed in vivo derived bovine embryos. Heifers were treated with oral propylene glycol for the last 20 days before embryo transfer (n = 142), and untreated as controls (n = 133). Progesterone, insulin, insulin-like growth factor-I, glucose, urea and triglyceride were analysed in blood on Day 0 and Day 7 of the estrous cycle corresponding to embryo transfer. The heifers were selected as recipients when showing progesterone levels <2.0 ng/ml (Day 0) and >2.5 ng/ml (Day 7), according to corpus luteum quality on Day 7 by technicians unaware of animals treated. Within treated animals, significantly more recipients were selected, and increased progesterone, corpus luteum quality, pregnancy and calving rates were recorded. Day 7 progesterone concentrations were higher in heifers treated and transferred. Propylene glycol increased insulin and insulin-like-growth factor-I, but glucose, urea and triglyceride did not vary. Furthermore, insulin-like-growth factor-I, glucose and triglyceride increased at estrous time, but urea decreased and insulin remained unaltered. Together with the sustained gain in pregnancy rates throughout the experiment (2 years), other evidences suggested that the observed effects did not rely on nutritional deficiency. Thus, propylene glycol improved pregnancy rates after embryo-transfer, and progesterone, insulin and insulin-like-growth factor-I are probably involved in this effect.

9-cis-retinoic acid during in vitro maturation improves development of the bovine oocyte and increases midkine but not IGF-I expression in cumulus-granulosa cells.

The isomer 9-cis of retinoic acid (9-cis-RA) exerts a beneficial effect on bovine in vitro development when added to in vitro maturation (IVM) culture. In the present work, 9-cis-RA 5 nM was found to be stimulatory as opposed to 500 nM (toxic). Cumulus-oocyte complexes (COCs) were treated with the found physiological dose 9-cis-RA 5 nM, and the next determinations performed: (1) relative expression of midkine (MK) and IGF-I, by reverse transcriptase-polymerase chain reaction (RT-PCR), in cumulus-granulosa cells detached from oocytes; (2) cytoplasmic granular migration, by labeling of oocytes with fluoroscein isothiocyanate lectins; and (3) in vitro survival of blastocysts after vitrification and warming. Gene expression of MK was enhanced by 9-cis-RA, but not by 1% ethanol (vehicle). However, we did not detect IGF-I expression, both in dependence on or in the absence of 9-cis-RA acting on cumulus-granulosa cells. The ability of vitrified blastocysts to survive in vitro was not improved by 9-cis-RA. Nevertheless, since only blastocysts obtained from oocytes matured with serum survived, more factors should be considered when evaluating cryopreservation survival. The complete granular migration observed in oocytes matured with 9-cis-RA anticipates the gain in developmental competence of the oocyte, being MK probably involved in this beneficial effect.

Use of two replacements of serum during bovine embryo culture in vitro.

This study evaluated the effect of two commercial serum replacements (Ultroser G and CPSR-3 on in vitro bovine embryo culture. In Experiment 1, zygotes were cultured in SOF+Ultroser G (2, 4 and 6%), SOF+CPSR-3 (2, 4 and 6%), and SOF+5% FCS (control). Blastocyst rates obtained after culturing with Ultroser G were lower than those with FCS. However, blastocyst rates for CPSR-3 were similar to those for serum. In addition, embryos produced in SOF+CPSR-3 had the same proportion inner cell mass number and total cell number as embryos cultured with FCS. In Experiment 2, a combination of serum replacements during different periods showed that treatment before the five-to eight-cell stages had no effect on further embryo development. However, treatments up to the morula stage affected blastocyst formation. The concentration of supplement and the timing of its inclusion in culture markedly affected embryo development. The serum replacement CPSR-3 can supplement embryo culture with blastocyst rates and quality similar to those for serum.

Macromolecular source as dependent on osmotic pressure and water source: effects on bovine in vitro embryo development and quality.

This study evaluated the protective effect of protein, as dependent on osmolarity, and the quality of water sources used to prepare embryo culture media. In Experiment 1, two concentrations of NaCl were used to obtain culture media with normal (280 mOSM) and low (245 mOSM) osmolarity, each supplemented with either bovine serum albumin (BSA) or polyvinyl alcohol (PVA). Low osmolarity improved blastocyst rates in the presence of BSA (P < 0.01) and tended to do it in medium containing PVA (P < 0.07). Furthermore, low osmolarity allowed PVA to increase inner cell mass (ICM) numbers and ICM/total cell rate (P < 0.05), while trophectoderm (TE) and total cell counts tended to decrease (P < 0.08). In Experiment 2, culture media were prepared with two water sources (Milli-Q and Sigma-W3500-) in combination with BSA or PVA. Both water sources yielded similar embryo development rates, but in the presence of BSA, Milli-Q water produced embryos with increased ICM/total cells rates (P < 0.05). On the contrary, Sigma water tended to increase trophectoderm cell counts (P < 0.08). In conclusion, the present study showed that low osmolarity is beneficial to embryo development and combinations of macromolecule and osmolarity influence trophectoderm differentiation. Both Milli-Q and Sigma supported embryo development at comparable rates, although in the presence of BSA, blastocysts obtained in the medium prepared with Milli-Q water had superior quality in terms of ICM/total cells rates.

Effects of acetoacetate and D-beta-hydroxybutyrate on bovine in vitro embryo development in serum-free medium.

It is known that the ketone bodies acetoacetate and D-beta-hydroxybutyrate can be metabolized by the early bovine embryo for in vitro development. In the present work, we report experiments leading to the culture of bovine embryos in the absence of serum. In vitro-produced bovine zygotes were cultured in modified synthetic oviduct fluid medium supplemented with acetoacetate derivatives, acetoacetate and D-beta-hydroxybutyrate. Acetoacetate and its derivatives prevented blastocysts from forming in the absence of serum during the whole culture period. However, from Days 6 to 8 of culture in the absence of serum, acetoacetate did not affect development as compared to controls containing lactate and pyruvate or no substrate. Interestingly, D-beta-hydroxybutyrate stimulated blastocyst and expansion development, and allowed lipid mobilization. In feeder cells coculture, embryos produced with D-beta-hydroxybutyrate showed improved hatching. Embryos cultured in D-beta-hydroxybutyrate were viable upon transfer to recipients, although no pregnancies were confirmed later by ultrasonic scanning. The protective effect of serum upon embryos cultured in medium containing acetoacetate is apparently not required in the presence of D-beta-hydroxybutyrate.