Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue

Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

Age-associated changes in cumulus cells and follicular fluid: The local oocyte microenvironment as a determinant of gamete quality

The ovary is the first organ to age in humans with functional decline evident already in women in their early thirties. Reproductive aging is characterized by a decrease in oocyte quantity and quality which is associated with an increase in infertility, spontaneous abortions, and birth defects. Reproductive aging also has implications for overall health due to decreased endocrinological output. Understanding the mechanisms underlying reproductive aging has significant societal implications as women globally are delaying childbearing and medical interventions have greatly increased the interval between menopause and total lifespan. Age-related changes inherent to the female gamete are well-characterized and include defects in chromosome and mitochondria structure, function, and regulation. More recently, it has been appreciated that the extra-follicular ovarian environment may have important direct or indirect impacts on the developing gamete, and age-dependent changes include increased fibrosis, inflammation, stiffness, and oxidative damage. The cumulus cells and follicular fluid which directly surround the oocyte during its final growth phase within the antral follicle represent additional critical local microenvironments. Here we systematically review the literature and evaluate the studies that investigated the age-related changes in cumulus cells and follicular fluid. Our findings demonstrate unique genetic, epigenetic, transcriptomic, and proteomic changes with associated metabolomic alterations, redox status imbalance, and increased apoptosis in the local oocyte microenvironment. We propose a model of how these changes interact, which may explain the rapid decline in gamete quality with age. We also review the limitations of published studies and highlight future research frontiers.

Effect of Some Months on Follicles and Oocytes Recovered from Iraqi Ewes

The aim of the current study was to know the effect of season on the numbers of follicles and Oocytes recovered during Several months. 304 genital systems of Ewes were collected from Al-Fallujah abattoir/ Al-Fallujah, Al-Anbar province, during the period from 3, January 2021 to 1, July 2021. The samples were transported with Cool Box contained normal saline to the Reproductive Biotechnology Lab/Dept. of Surgery and Obstetrics / College of Vet. Medicine, University of Fallujah. The results showed that the total numbers of Oocytes with Cumulus cells recovered were 1037 oocytes. The results also showed a significant difference (P≤ 0.01) in the numbers of follicles between the right and the left ovaries. The numbers of follicles present at right ovaries were 776 in a percentage of (56.73%) from the total numbers where it was 592 follicles at the left ovaries in a percent of (43.27%) from the total numbers. It has been observed superiority of right ovary on the left ovary in the numbers of oocytes recovered, where it was 603 oocytes (58.15 %) from the right ovary and 434 oocytes (41.85 %) from the Left ovary. Also, the results showed a high Significant difference (P≤ 0.01) in the size of large and small follicles numbers. when the small follicles have large numbers. It has been also concluded from this study that there was a significant difference (P≤ 0.01) in the numbers of follicles and oocytes between the months of March and April as compared with other months.

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically β-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Production, Evaluation, Cultivation and In vitro Fertilization of Cattle Oocytes: Recent Trends in Reproductive Biotechnologies in Animal Breeding : A Review

The article presents the results of assessing reproductive biotechnology for cattle breeding. The issues of obtaining genetic material from bulls-producers and oocytes from donor cows, for their further cultivation and obtaining embryos, in order to replicate highly productive offspring from valuable animals, are considered. Oocyte production was analyzed in three different ways: puncture, section, and aspiration. A total number of 156 oocyte-cumulus complexes (OCCs) were collected out of 40 ovaries by puncture – 50 pcs, section – 47 pcs, and aspiration – 59 pcs. The results showed that puncture and section gave significantly higher total OCCs per an ovary (4.16 and 4.0, respectively) than aspiration (3.68), but a higher number of normal (grade A and B) OCCs per an ovary was observed with aspiration (2.5) than with puncture (1.82) and section (2.00). During aspiration, oocyte-cumulus complexes were collected from the surface of follicles with a diameter of 3 to 8 mm using a needle. During puncture, all surfaces were pierced with a hypodermic needle; during section, incisions were made along the entire ovarian surface with a scalpel, that is, all sizes of superficial follicles were collected. OCCs were divided into 4 classes based on cumulus and nucleus cells: grade A – oocytes completely surrounded by cumulus cells; Grade B – oocytes partially surrounded by cumulus cells; Grade C – oocytes not surrounded by cumulus cells; and grade D – degeneration observed in both oocyte and cumulus cells. Grades A and B were considered normal, while grades C and D were considered to be damaged. The obtained oocytes were cultured and fertilized in vitro, which means that their fertilization with sperm occurred under artificially maintained optimal conditions outside the body. Normal fertilization results are zygote formation with male and female pronuclei (PN). As a result of the conducted fertilization of 251 mature oocytes, 142 fertilizations were successful, which amounted to 56.57%.