Follicular size, follicular atresia, and oocyte morphology were investigated for the possible relation of these characteristics to the developmental competence of bovine oocytes. Ovaries from a local slaughterhouse were dissected to obtain a heterogeneous population of follicles. Half of each follicle was fixed for histological analysis, and the oocytes were detached carefully and cultured individually. Before in vitro maturation, the oocytes were grouped into six different classes based on the morphology of the cumulus and the ooplasm: classes 1 and 2 represent oocytes with a homogeneous ooplasm plus a compact and complete cumulus, and classes 3-6 represent oocytes with a granulated ooplasm and an incomplete and/or expanded cumulus. Oocytes from class 3 (beginning of expansion in outer cumulus layers and slight granulations in the ooplasm) developed past the 16-cell stage significantly (P < 0.05) more than oocytes with a compact and complete cumulus (classes 1 and 2) and oocytes from classes 4-6 (incomplete and/or expanded cumulus) after 5 days of in vitro culture. Oocytes from follicles measuring 3 mm or less did not develop past the 16-cell stage, whereas follicles of 3-5 mm and 5 mm or larger developed at similar rates (17% and 21% morulae, respectively). The state of the follicle did not affect whether an embryo reached at least the 16-cell stage, as comparable rates were obtained in all three groups of follicles: nonatretic (20%), intermediate (14%), and slightly atretic (16%).(ABSTRACT TRUNCATED AT 250 WORDS)

Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes

DNA damage in the male germ line has been linked with a variety of adverse clinical outcomes including impaired fertility, an increased incidence of miscarriage and an enhanced risk of disease in the offspring. The origins of this DNA damage could, in principle, involve: (i) abortive apoptosis initiated post meiotically when the ability to drive this process to completion is in decline (ii) unresolved strand breaks created during spermiogenesis to relieve the torsional stresses associated with chromatin remodelling and (iii) oxidative stress. In this article, we present a two-step hypothesis for the origins of DNA damage in human spermatozoa that highlights the significance of oxidative stress acting on vulnerable, poorly protaminated cells generated as a result of defective spermiogenesis. We further propose that these defective cells are characterized by several hallmarks of 'dysmaturity' including the retention of excess residual cytoplasm, persistent nuclear histones, poor zona binding and disrupted chaperone content. The oxidative stress experienced by these cells may originate from infiltrating leukocytes or, possibly, the entry of spermatozoa into an apoptosis-like cascade characterized by the mitochondrial generation of reactive oxygen species. This oxidative stress may be exacerbated by a decline in local antioxidant protection, particularly during epididymal maturation. Finally, if oxidative stress is a major cause of sperm DNA damage then antioxidants should have an important therapeutic role to play in the clinical management of male infertility. Carefully controlled studies are now needed to critically examine this possibility.

On the possible origins of DNA damage in human spermatozoa

Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization.

Theca cells function in a diverse range of necessary roles during folliculogenesis; to synthesize androgens, provide crosstalk with granulosa cells and oocytes during development, and provide structural support of the growing follicle as it progresses through the developmental stages to produce a mature and fertilizable oocyte. Thecal cells are thought to be recruited from surrounding stromal tissue by factors secreted from an activated primary follicle. The precise origin and identity of these recruiting factors are currently not clear, but it appears that thecal recruitment and/or differentiation involves not just one signal, but a complex and tightly controlled combination of multiple factors. It is clear that thecal cells are fundamental for follicular growth, providing all the androgens required by the developing follicle(s) for conversion into estrogens by the granulosa cells. Their function is enabled through the establishment of a vascular system providing communication with the pituitary axis throughout the reproductive cycle, and delivering essential nutrients to these highly active cells. During development, the majority of follicles undergo atresia, and the theca cells are often the final follicular cell type to die. For those follicles that do ovulate, the theca cells then undergo hormone-dependent differentiation into luteinized thecal cells of the corpus luteum. While the theca is an essential component of follicle development and ovulation, we do not yet fully understand the control of recruitment and function of theca cells, an important consideration since their function appears to be altered in certain causes of infertility.

/pdf/theca-the-forgotten-cell-of-the-ovarian-follicle-557ura2rt1.pdf

Theca: the forgotten cell of the ovarian follicle.

There has been an improvement in the blastocyst rates achieved following in-vitro embryo production that can largely be attributed to improved embryo culture conditions based on an increased knowledge of the in-vivo environment, as well as the metabolic needs of the embryo. Despite this, in-vitro oocyte maturation (IVM) conditions have remained largely unchanged. Within the antral follicle, numerous events affect oocyte maturation and the acquisition of developmental competency, including: interactions between somatic cells of the follicle (in particular cumulus cells) and the oocyte; the composition of follicular fluid; and the temperature and vascularity of the follicular environment. Many of these factors change with follicle size and oocyte growth. In contrast, culture conditions for IVM are based on somatic cells that often do not reflect the follicular environment, and/or have complex compositions or additives such as macromolecule supplements that are undefined in nature. Metabolites included in media such as glucose, pyruvate, oxygen and amino acids have been shown to have differential influences on oocyte maturation and competency. Manipulation of these factors and application of gained knowledge of the in-vivo environment may result in improved in-vitro oocyte maturation and overall in-vitro embryo production.

/pdf/effects-of-in-vivo-and-in-vitro-environments-on-the-14ixyv9kff.pdf

Effects of in-vivo and in-vitro environments on the metabolism of the cumulus–oocyte complex and its influence on oocyte developmental capacity

Developmental competence of pig oocytes matured and fertilized in vitro

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small ( 5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

https://bioone.org/journals/Biology-of-Reproduction/volume-63/issue-3/biolreprod63.3.858/Vascular-Endothelial-Growth-Factor-Production-in-Growing-Pig-Antral-Folliclesa/10.1095/biolreprod63.3.858.pdf

Vascular Endothelial Growth Factor Production in Growing Pig Antral Follicles

In order to investigate the effect of the somatic cells of the follicle on the preparation of the oocyte for fertilization three experiments were carried out. In the first, pig oocytes, cultured for 46 h in the presence of extroverted follicles (follicle oocytes) or surrounded by the cumulus (cumulus oocytes), were exposed to sperm in an in vitro fertilization system. Follicle oocytes showed a higher rate of fertilization than that recorded in cumulus oocytes (80% vs. 47%). In addition, significantly more sperm penetrated into the ooplasm of follicular oocytes (3.77/egg) than into that of cumulus oocytes (1.42/egg). To investigate the reason for the observed fertilization difference zona-free oocytes were studied in the second experiment. Significantly more spermatozoa were recorded in the ooplasm of follicle oocytes than in that of cumulus oocytes, thus suggesting that the effect of the follicle on fertilizability may partly depend on an action on the plasma membrane of the oocyte. A further effect of the follicular tissue was on cytoplasmic maturation: only follicular oocytes were capable of consistently promoting male pronucleus formation. In cumulus oocytes, sperm that entered the cytoplasm remained in a condensed form near the female pronucleus. In the third experiment cumulus oocytes and denuded oocytes were matured in medium that had been previously used to mature follicle oocytes. This conditioned medium was alone able to affect sperm penetration and male pronucleus formation in cumulus oocytes, but it did not exert any influence on denuded oocytes. This suggests that the observed effect of the follicle is mediated by soluble factors that, however, cannot influence the oocyte without some direct cell-oocyte contract.

Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation.

The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (,4 mm), medium (4‐5 mm), and large (.5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (,3 ng/ml), high in large follicles (.10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% 6 0.58% vs. 1.29% 6 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150‐300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic mediumsize follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

https://bioone.org/journals/biology-of-reproduction/volume-65/issue-4/biolreprod65.4.1014/Follicle-Activation-Involves-Vascular-Endothelial-Growth-Factor-Production-and-Increased/10.1095/biolreprod65.4.1014.pdf

Follicle Activation Involves Vascular Endothelial Growth Factor Production and Increased Blood Vessel Extension

In order to investigate whether the follicular tissue influences cumulus-oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus-enclosed pig oocytes were cultured for 44 h in control medium (modified TCM-199) or in follicle-conditioned medium, and the intercellular coupling was studied by measuring 3H-uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24-32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24-32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45-kD protein band, while those maturing in control medium were identifiable by a marked 56-kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17 beta-estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei. These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.

Giovanna Galeati

Papers

Developmental competence of pig oocytes matured and fertilized in vitro

Vascular Endothelial Growth Factor Production in Growing Pig Antral Follicles

Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation.

Follicle Activation Involves Vascular Endothelial Growth Factor Production and Increased Blood Vessel Extension

Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte.