Michel Franck

Normal pregnancies and live births after autograft of frozen-thawed hemi-ovaries into ewes

OBJECTIVE To evaluate long-term outcome of autotransplantation of cryopreserved hemi-ovaries into ewes. DESIGN Animal study. SETTING University fertility center, Hospices Civils de Lyon; and Ecole Nationale Vétérinaire de Lyon. PATIENT(S) Grivette ewes. INTERVENTION(S) Six hemi-ovaries from 6 ewes aged 6 to 12 months were frozen with a slow cooling protocol using 2 M of dimethyl sulfoxide as cryoprotectant. After dissection of the medulla, the hemi-ovarian cortex was stored at -196 degrees C in liquid nitrogen. Freezing procedure was performed with a programmable freezer. Semiautomatic seeding was performed before crystallization. Four to 6 weeks after the first laparotomy, the left ovary was removed and the frozen-thawed hemi-ovary was sutured. MAIN OUTCOME MEASURE(S) Mean plasma concentrations of FSH, LH, and progesterone after autotransplantation of frozen-thawed hemi-ovary. Ultrasonography was done to confirm pregnancy. Blood samples were collected weekly to measure FSH, LH, and progesterone. After the first birth, the autografted ovary was removed for histologic examination. RESULT(S) Plasma progesterone concentration increased in a regular manner in all ewes except one 4 weeks after the graft. Concentrations of FSH and LH did not reach the menopausal level. Four pregnancies occurred, from which 6 lambs were born. The first delivery of a normal lamb occurred after 135 days of gestation; the lamb died immediately after birth. The second delivery of two normal lambs occurred after 130 days of gestation. A caesarean section was performed on the third pregnant ewe the 110th days of gestation because the ewe had a vaginal prolapsus. The two normal lambs and the ewe died after surgery. The fourth birth of a normal lamb occurred after 132 days of gestation. Histologic examination of the grafted frozen-thawed ovary showed a regressing corpus luteum and few primordial and antral follicles. CONCLUSION(S) These four pregnancies in a ewe model may indicate that women who undergo preservation of their ovaries before chemotherapy or radiotherapy can have successful pregnancy.

Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols

OBJECTIVE To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. SETTING Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France. ANIMAL(S) Lambs 5 to 6 months of age. INTERVENTION(S) Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. MAIN OUTCOME MEASURE(S) Follicular mortality and histologic structure. RESULT(S) For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S) Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.

Long-term follow-up of cryopreserved hemi-ovary autografts in ewes: pregnancies, births, and histologic assessment

OBJECTIVE To evaluate a 2-year follow-up of cryopreserved hemi-ovary autografts in ewes. DESIGN Animal study. SERTTING: University fertility center, Hospices Civils de Lyon; Ecole Nationale Vétérinaire de Lyon, INSERM U 418 Hocaron;pital Debrousse, Lyon; and Hôpital Edouard Herriot, Lyon, France. PATIENT(S) Grivette ewes. INTERVENTION(S) Recently we reported four pregnancies and six live births after transplantation of frozen-thawed hemi-ovary in six different ewes. The four remaining ewes were monitored for 2 years. After the last birth, the autografted ovary was removed in each ewe during a final laparotomy. The entire grafted ovary was sliced to estimate the remaining primordial follicle population 2 years after grafting. MAIN OUTCOME MEASURE(S) Uterine ultrasound scanning was performed to diagnose pregnancy. Histological assessment of the grafted ovary was performed after delivery. RESULT(S) The four remaining ewes began new gestations. For two of them, this was a second gestation obtained more than 2 years after the autograft. These two ewes delivered male lambs, which died immediately after delivery because of distocia. The lambs were both oversized for gestational age; autopsy found no malformation. A twin pregnancy of a healthy male and a healthy female occurred in May 2002, and a singleton male was born in February 2002. All grafted ovaries showed drastic reduction in follicle population. CONCLUSION(S) Frozen-thawed ovary autograft allowed recovery of fertility a very long time after the procedure despite a drastic reduction in the total number of follicles.

Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification

OBJECTIVE To evaluate recovery of endocrine function and fertility after transplantation and vitrification of whole ovaries. DESIGN Animal study. SETTING Lyon Veterinary School, France. ANIMAL(S) Ewes. INTERVENTION(S) In group 1 (n = 5), the left ovary was removed with its vascular pedicle and was transplanted onto the contralateral pedicle. In group 2 (n = 5), the left ovary with its pedicle was cryopreserved after a vitrification procedure. After thawing, transplantation was performed by microvascular anastomosis to the contralateral ovarian pedicle. MAIN OUTCOME MEASURE(S) Median ischemia time, progesterone levels, histologic examination. RESULT(S) Successful microsurgical transplantation was performed in both groups. The median ischemia time was statistically significantly longer in group 2 (287 minutes, range: 226 to 349] versus 129 minutes [range: 125 to 130]) in group 1. In group 1, four sheep recovered spontaneous ovarian endocrine function about 2.5 (range: 2.00 to 3.75) months after transplantation. Two ewes gave healthy live births at 12 and 25 months, respectively, after transplantation. In group 2, one ewe recovered ovarian endocrine function 6 months after transplantation. However, histologic evaluation showed a follicular survival rate of 6% in group 1, and total follicle loss in group 2. CONCLUSION(S) Autograft of whole sheep ovaries with microvascular anastomosis seems technically feasible but resulted in a very poor follicle survival rate (6%), in spite of endocrine function recovery and birth of two lambs. Attempts at cryopreservation with vitrification resulted in no follicle survival at all.

Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep

OBJECTIVE To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols. DESIGN Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. SETTING Fertility clinic in a large university hospital. ANIMALS Five- to 6-month-old lambs. INTERVENTION(S) Two-millimeter-thick slices of hemi-ovary cortex were prepared. MAIN OUTCOME MEASURE(S) Histological structure and DNA fragmentation. RESULT(S) In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% +/- 13.2% vs. 68.4% +/- 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group. CONCLUSION(S) The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.

Characterisation of PSE zones in semimembranosus pig muscle.

Pig semimembranosus muscles, sampled from normal hams or from PSE-zones of defective hams, were analysed by histochemistry and electrophoretic techniques. PSE zones were characterised by a disorganisation of fibre alignment and a significant increase of inter fibre spacing (26.2% vs. 16.9%, p<0.05). Protein solubility was significantly lower in defective muscle (55.4 vs. 91.5mg/g, p<0.001). SDS-PAGE evidenced in such samples a lower abundance of the 97, 40 and 26kDa bands in the sarcoplasmic fraction and a higher abundance of the 97, 58, 34, 31, 15 and 11kDa bands in the myofibrillar fraction. Intensity of the MHC band (200kDa) was lower in PSE zone samples. By 2-D electrophoresis, it was shown that troponin T, MLC 1 and alpha-crystallin were less proteolysed in defective muscles, while creatine kinase fragments were more represented. One form of HSP 27 was absent from PSE zone samples. Overall, meat from PSE-zones and fast pH fall-PSE meat show numerous histological and biochemical similarities, particularly in their protein characteristics.

The cryopreservation of ovarian tissue: uses and indications in veterinary medicine

Animal experiments have shown that cryopreservation of the ovarian cortex, containing primordial follicles, could be used to preserve gametes thereby restoring fertility in humans and animals. During the last 100 years, many hundreds of species have been lost, and a third of the breeding animals are threatened with extinction. To preserve genetic diversity, notably for the conservation of endangered species, it is essential to conserve female and male gametes. Today, biotechnologies such as artificial insemination and embryo transfer are used in breeding programs and are well developed. However, even using these advanced techniques, there are problems due to the limited number of individuals used as the source of gametes, so that the risk of inbreeding is high, even in large populations. To preserve genetic diversity, it is necessary to create gene banks of male and female gametes and embryos, using a very large number of individual donors. Cryopreservation of ovarian tissue could present a means for enlarging the gene pool. Cryopreserved ovarian tissue could be used in auto- or xenografts, or for in vitro maturation (IVM) of primordial follicles. In this review, we describe the processes for cryopreservation of ovarian tissue and the various possibilities for using it.

Identification of five chromosomal regions involved in predisposition to melanoma by genome-wide scan in the MeLiM swine model.

In human familial melanoma, 3 risk susceptibility genes are already known, CDKN2A, CDK4 and MC1R. However, various observations suggest that other melanoma susceptibility genes have not yet been identified. To search for new susceptibility loci, we used the MeLiM swine as an animal model of hereditary melanoma to perform a genome scan for linkage to melanoma. Founders of the affected MeLiM stock were crossed with each other and with healthy Duroc pigs, generating MeLiM, F1 and backcross families. As we had previously excluded the MeLiM CDKN2A gene, we paid special attention to CDK4 and MC1R, as well as to other candidates such as BRAF and the SLA complex, mapping them on the swine radiation hybrid map and/or isolating close microsatellite markers to introduce them into the genome scan. The results revealed, first, that swine melanoma was inherited as an autosomal dominant trait with incomplete penetrance, preferably in black animals. Second, 4 chromosomal regions potentially involved in melanoma susceptibility were identified on Sus Scrofa chromosomes (SSC) 1, 2, 7 and 8, respectively, in intervals 44–103, 1.9–18, 59–73 and 47–62 cM. A fifth region close to MC1R was revealed on SSC 6 by analyzing an individual marker located at position 7.5 cM. Lastly, CDK4 and BRAF were unlikely to be melanoma susceptibility genes in the MeLiM swine model. The 3 regions on SSC 1, 6 and 7, respectively, have counterparts on human chromosomes (HSA) 9p, 16q and 6p, harboring melanoma candidate loci. The 2 others, on SSC 2 and 8, have counterparts on HSA 11 and 4, which might therefore be of interest for human studies.

Brain pericytes from stress-susceptible pigs increase blood-brain barrier permeability in vitro

BackgroundThe function of pericytes remains questionable but with improved cultured technique and the use of genetically modified animals, it has become increasingly clear that pericytes are an integral part of blood–brain barrier (BBB) function, and the involvement of pericyte dysfunction in certain cerebrovascular diseases is now emerging. The porcine stress syndrome (PSS) is the only confirmed, homologous model of malignant hyperthermia (MH) in veterinary medicine. Affected animals can experience upon slaughter a range of symptoms, including skeletal muscle rigidity, metabolic acidosis, tachycardia and fever, similar to the human syndrome. Symptoms are due to an enhanced calcium release from intracellular stores. These conditions are associated with a point mutation in ryr1/hal gene, encoding the ryanodine receptor, a calcium channel. Important blood vessel wall muscle modifications have been described in PSS, but potential brain vessel changes have never been documented in this syndrome.MethodsIn the present work, histological and ultrastructural analyses of brain capillaries from wild type and ryr1 mutated pigs were conducted to investigate the potential impairment of pericytes, in this pathology. In addition, brain pericytes were isolated from the three porcine genotypes (wild-type NN pigs; Nn and nn pigs, bearing one or two (n) mutant ryr1/hal alleles, respectively), and tested in vitro for their influence on the permeability of BBB endothelial monolayers.ResultsEnlarged perivascular spaces were observed in ryr1-mutant samples, corresponding to a partial or total detachment of the astrocytic endfeet. These spaces were electron lucent and sometimes filled with lipid deposits and swollen astrocytic feet. At the ultrastructural level, brain pericytes did not seem to be affected because they showed regular morphology and characteristics, so we aimed to check their ability to maintain BBB properties in vitro. Our results indicated that pericytes from the three genotypes of pigs had differing influences on the BBB. Unlike pericytes from NN pigs, pericytes from Nn and nn pigs were not able to maintain low BBB permeability.ConclusionsElectron microscopy observations demonstrated brain capillary modifications in PSS condition, but no change in pericyte morphology. Results from in vitro experiments suggest that brain pericytes from ryr1 mutated pigs, even if they are not affected by this condition at the ultrastructural level, are not able to maintain BBB integrity in comparison with pericytes from wild-type animals.

Updated estimates of HAL n and RN— effects on pork quality: Fresh and processed loin and ham

A 1000-pig F2 intercross QTL detection experimental population was generated using two commercial sire lines. Independent carriers of HAL n and RN- mutations (10% and 14%, respectively) were included in this population as control genotypes. The effects of HAL n and RN- heterozygous genotypes on fresh and transformed loins and hams were estimated using a mixed model methodology. The results document the unfavorable effects of both mutations on meat quality. Smaller effects of HAL Nn genotype compared to HAL nn or RN-rn+ genotypes were estimated. Interestingly, effects of HAL Nn genotype on meat pH and loin color could be insignificant at 24-h postmortem, but translate into higher water losses on storage and cooking, and result in tougher cooked loin. Using the same methodology, significant effects of the PRKAG3 (RN) I199 allele on ultimate pH values but not on glycolytic potential were observed.