The morphological characteristics of frozen-thawed human mature oocytes (n = 12) were studied by light and transmission electron microscopy following cryopreservation using a slow cooling protocol including increasing concentrations of ethylene glycol (0.5-1.5 mol/l) and sucrose 0.2 mol/l in the freezing solution. Fresh human mature oocytes (n = 12) were used as controls. Fresh and frozen-thawed oocytes appeared rounded in section, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Disorganization of mitochondria-smooth endoplasmic reticulum aggregates and a decreased complement of microvilli and cortical granules were frequently observable in frozen-thawed oocytes. Increased density of the inner zona pellucida, possibly related to the occurrence of zona 'hardening', was sometimes found associated with a reduced amount of cortical granules. In addition, delamination of the zona pellucida was evident in some frozen-thawed samples. Finally, numerous vacuoles and secondary lysosomes were detected in the ooplasm of most frozen-thawed oocytes. In conclusion, frozen-thawed oocytes treated with ethylene glycol may show a variety of ultrastructural alterations, possibly related, at least in part, to the use of this cryoprotectant. Thus, the ethylene glycol-based protocol of slow cooling herein described does not seem to offer significant advantages in terms of oocyte structural preservation.
Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.
The possible acute morphological changes induced by electrical transcranial unifocal stimulation (eTCS) in the rabbit extracerebral tissues were studied by light and scanning electron microscopy. In order to do this, a wide range of electric stimuli with respect to those employed in the clinical practice were utilized. Either surface electrodes were attached to the scalp, or needle electrodes were infixed in the subcutaneous tissue. Beneath the cathode a blood extravasation was constantly observed in the subcutaneous tissue of the scalp; the different electrode arrays produced either a large hemorrhagic lesion or a few petechiae. Beneath the anode, the damage was limited to the scalp, or reached the meninges when stimuli longer than 0.2 ms were used. Irrespective of the electrode arrays, the scalp and the dura mater displayed hemorrhagic petechiae over a limited area about 2–3 mm in extent. Moreover, the leptomeningeal membrane was microscopically disrupted over an area less than 1 mm large; therein the squamous, overlapping cells were transformed into fusiform or macrophage-like cells. Unduly intense eTCS produces evident hemorrhagic lesions in the scalp and in the dura mater, whereas it induces microscopic, reactive changes in the leptomeninx.
The ovarian function during reproductive life is related to a normal development of blood vessel neoformation and consequently to the expression of several factors that may upregulate/downregulate follicle and corpus luteum angiogenesis [1,2]. Follicle activation involves vascular endothelial growth factor production and increases blood vessels extension. Numerous morphological studies, mainly performed by means of vascular casting media, analysed the distribution and cyclical rearrangement of ovarian blood vessels in several mammals, in different experimental conditions [3]. Despite the wide literature on the blood vessel distribution in the mammalian ovary, only a few information is available on ovarian angiogenic morphological aspects of developing and atretic follicles, especially in domestic animals, that shows morphofunctional characteristics of the ovarian follicle more similar to humans than rodent models. In addition, it is not yet clear which morphological aspects are related to a correct angiogenesis and capillary regression, especially when evaluated in the light of species differences. Therefore, in order to better understand the morphological mechanisms regulating the microvascular changes occurring in the theca layer before ovulation of larger follicles, we have reviewed by scanning electron microscopy (SEM) of vascular corrosion casts (VCC) the ovarian microvasculature in different experimental conditions, in rodents and domestic animals.Rat, rabbit, pig, sheep, and cow ovaries, at different reproductive stages and or/subjected to different protocols of hormonal stimulation were studied. Rat model was used as control. Rabbit was chosen as a model for reflex ovulation [4]; pig [2,5,6] and sheep [7] as multiovulatory species and cow [8] as a monovulatory model similar to humans. SEM of CC was adopted because it is one of the technique of choice for 3D visualization of blood vessels and capillary network arrangements [4]. Methods and hormonal protocols were previously described [2,[4][5][6][7][8][9]].An intensive functional and morphological remodeling of cortical venules and arterioles accompanied follicle development [9]. Follicle angiogenesis was demonstrated during follicular maturation in all species studied [3]. SEM of VCC demonstrated the differentiation of several microstructures, namely buds, sprouts (sprouting angiogenesis) and intussusceptions (non sprouting angiogenesis) that allowed firstly the growth, then the duplication and multiplication of the preexisting capillaries. The gradual formation of a dense sinusoidal network supplying the theca layer of fully developed follicles (dominant) was also clearly shown. Atretic and subordinated follicles presented degenerative microvascular structures such as thinned capillaries and avascular areas. Species differences were related to follicle size and to mechanisms of follicle selection. In rodents, significant angiogenic morphological figures were mainly confined to the capillaries of the inner thecal layer. In larger follicles of domestic ani...
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