Follicle Classes Research Articles

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Follicle activation and 'burn-out' contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness

What are the effects of thin ovarian grafts compared with grafts of the standard thickness on follicle loss post-transplantation? Transplantation of reduced-thickness ovarian grafts led to intense activation and 'burn-out' a short time after transplantation resulting in significant folllicle loss. Transplantation of fresh and frozen-thawed ovarian tissue has been proved successful, but techniques vary and are not optimised, often resulting in significant follicular loss. Follicle loss is mostly related to the freezing-thawing process and to post-transplantation hypoxia. Bovine ovarian tissue strips (n = 55) were prepared in two groups of conventional-thickness strips (1-2 mm) or thin strips (0.5-0.9 mm). Fresh or frozen-thawed samples were xenotransplanted into sterilized immune-deficient mice (n = 49). Non-transplanted conventional size fresh samples were used as controls (n = 6). Grafts from all study groups were recovered after 7 days for analysis. Morphometric differential counting of follicle classes was performed by two observers. Immunohistochemistry was conducted for proliferation (Ki67), cortical fibrosis (Masson tri-chrome) and blood-vessel density (CD31). Results were expressed as the mean number of dormant or growing follicle (GF) type per section or total follicle counts per graft. Blood-vessel density was calculated per mm(2). P-values <0.05 were considered statistically significant. The loss of all follicle types, and most noteably of primordial follicles (PMFs), was observed 7 days post-transplantation (P < 0.05). The relatively high number of GFs and the positive Ki67 staining in all recovered grafts indicated that follicle activation was depleting the resting follicle pool. The reduced graft thickness had an adverse effect on the number of recovered follicles, especially on the resting non-GFs in the fresh, and more so in the frozen-thawed, samples (P < 0.05). Extensive stromal fibrosis and high blood-vessel density were observed in all grafts with no advantage in the thin prepared grafts. This study used only one species of ovaries (bovine) for xenotransplantation. The immediate post-transplantation events were not visualized directly nor were the molecules involved in follicle activation studied. Follicle activation and 'burn-out' appear to be important in follicle loss after transplantation. Reducing graft thickness in an attempt to improve freezing conditions and reduce post-transplantation ischemia has adverse effects on the graft follicle pool due to increased activation and loss. Agents which prevent 'burn-out' will potentially improve follicle pool survival. This study was funded by research grants from the Israeli Science Foundation (No. 1675/10), the Israeli Jack Craps foundation and the Israel Cancer Research Fund (ICRF No. 12-3081). The authors have no competing interest to declare.

The two classes of primordial follicles in the mouse ovary: their development, physiological functions and implications for future research

Ovarian follicles are the basic functional units in the mammalian ovary. This review summarizes early pioneering studies and focuses on recent progress that has shown that there are two distinct classes of primordial follicles in the ovary: the first wave of primordial follicles that are activated immediately after they are formed and the adult primordial follicles that are activated gradually in later life. These two separate classes have been proposed for two decades, but sufficient experimental evidence to support this hypothesis has only been obtained recently using newly developed follicular tracing techniques in genetically modified mouse models. These two follicle populations differ from each other primarily in terms of their developmental dynamics and their contributions to ovarian physiology. It is apparent now that these two follicle populations should be treated separately, and such knowledge will hopefully lead to a more in-depth understanding of how distinct types of primordial follicles contribute to physiologic and pathologic alterations of the mammalian ovary.

Changes in granulosa cells gene expression associated with growth, plateau and atretic phases in medium bovine follicles.

BackgroundThe objective of this study was to build the transcriptomic profile of granulosa cells originating from follicles 6 to 9 mm in diameter in dairy cattle using microarrays.MethodsGranulosa cells originating from three different phases of antral follicle growth were compared: growing (G), plateau (P) and atresia (A), as categorized by flow cytometry profiles of DNA. The growing and atretic conditions were each hybridized against the plateau condition as a reference in order to understand the specific biological mechanisms modulated in this class of follicles.Results2,942 genes were differentially expressed (P < 0.05) in P vs. G and 1,974 in A vs. P. A clear segregation of the 3 phases was confirmed by between group analysis (BGA). The first characteristic of the plateau phase is the activation of the upstream regulators TP53 and PTEN which participate in the reduction of cell growth through MYC, FOS and E2F1-2-3. We also observed the down-regulation of steroidogenesis genes: CYP11A1 and CYP19A1, in the granulosa cells of the plateau phase relative to the growth phase. On the other hand, the A vs. P contrast showed up-regulation of multiple transcripts associated to apoptosis: CCT2, DAB2, DSG2 and TGM2.ConclusionsThis study offers multiple candidate genes to be further studied in order to elucidate their role in the modulation of follicular development and, ultimately, of oocyte quality.

Fuzzy inference system for follicle detection in ultrasound images of ovaries

The ovarian ultrasound imaging is an effective tool in infertility treatment. Monitoring the follicles is especially important in human reproduction. Periodic measurements of the size and shape of follicles over several days are the primary means of evaluation by physicians. Today monitoring the follicles is done by non-automatic means with human interaction. This work can be very demanding and inaccurate and, in most of the cases, means only an additional burden for medical experts. To improve the performance of follicle detection in ultrasound images of ovaries, we develop a new algorithm using fuzzy logic. The proposed method employs contourlet transform for despeckling the ultrasound images of ovaries, active contours without edge method for segmentation and fuzzy logic for classification. The follicles in an ovary are characterized by seven geometric features which are used as inputs to the fuzzy logic block of the Fuzzy Inference System. The output of the fuzzy logic block is a follicle class or non follicle class. The fuzzy-knowledge-base consists of a set of physically interpretable if-then rules providing physical insight into the process. The experimentation has been done using sample ultrasound images of ovaries and the results are compared with the inferences drawn by interval based classifier and also those drawn by the medical expert. The experimental results demonstrate the efficacy of the proposed method.

Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions

In the mammalian ovary, progressive activation of primordial follicles serves as the source of fertilizable ova, and disorders in the development of primordial follicles lead to various ovarian diseases. However, very little is known about the developmental dynamics of primordial follicles under physiological conditions, and the fates of distinct populations of primordial follicles also remain unclear. In this study, by generating the Foxl2-CreERT2 and Sohlh1-CreERT2 inducible mouse models, we have specifically labeled and traced the in vivo development of two classes of primordial follicles, the first wave of simultaneously activated follicles after birth and the primordial follicles that are gradually activated in adulthood. Our results show that the first wave of follicles exists in the ovaries for ∼3 months and contributes to the onset of puberty and to early fertility. The primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life. Moreover, by tracing the time periods needed for primordial follicles to reach various advanced stages in vivo, we were able to determine the exact developmental dynamics of the two classes of primordial follicles. We have now revealed the lifelong developmental dynamics of ovarian primordial follicles under physiological conditions and have clearly shown that two classes of primordial follicles follow distinct, age-dependent developmental paths and play different roles in the mammalian reproductive lifespan.

Biochemical and steroid concentrations in follicular fluid and blood plasma in different follicular waves of the estrous cycle from normal and superovulated beef cows

The objectives of the current study were to (i) define the changes in size and number of follicles populations, (ii) determine the follicular fluid (FF) biochemical and steroid concentrations collected from different-sized follicles (5–9 and ≥10mm) and (iii) compare between biochemical and hormonal concentrations of FF with those in blood plasma in relation to the first two follicular waves of the estrous cycle (days 4 and 13) from normal and cows primed for superovulation. After estrus, cows (n=20) were assigned randomly to each of four treatment groups. Group 1: ovariectomy on day 4 (day 0=ovulation). Group 2: FSH treatment and ovariectomy on day 4. Group 3: dominant follicle ablation (DFA) on day 8 and ovariectomy on day 13. Group 4: DFA on day 8, FSH treatment and ovariectomy on day 13. Blood samples were collected and FF was aspirated and pooled per follicle class within cow to determine glucose, urea, triglycerides, cholesterol, total protein, albumin, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transpeptidase, aspartate aminotransferase, creatin phosphokinase, estradiol-17β and progesterone concentrations. Follicular class×follicular wave interaction was detected for albumin and lactate dehydrogenase. Results showed that FF concentrations of cholesterol increased from medium to large follicles and decreased for urea and aspartate aminotransferase. Tryglycerides and total protein were greater in the second than in the first follicular wave. FSH treatment decreased FF alkaline phosphatase, E2 and P4 concentrations. Quantitative differences between these fluids are discussed with respect to follicular development.

In vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method

The objective was to conduct a preliminary evaluation of the efficacy of two media for in vitro culture of equine preantral follicles. Ovarian cortical strips were obtained from mares (N = 10) via the Biopsy Pick-Up method during the breeding season. Ovarian tissue was immediately submitted to histological analysis (noncultured control; D0) or cultured in situ for 1 day (D1) or 7 days (D7) in either α-MEM or TCM-199 and submitted to histological analysis, generating five treatment groups: noncultured control, α-MEM:D1, TCM-199:D1, α-MEM:D7, and TCM-199:D7. Preantral follicles were evaluated for follicle class (primordial, transitional, primary, and secondary) and morphology (normal vs. abnormal). A total of 142 preantral follicles were analyzed in five replicates. No follicles were observed in the TCM-199:D7 treatment group. The proportion of primordial follicles was higher (P < 0.03) in the control compared to the α-MEM:D7 treatment group. The proportion of primary follicles was higher (P < 0.04) in the α-MEM:D7 treatment group compared to the control. The proportion of developing follicles (transitional, primary, and secondary) was higher (P < 0.03) in the α-MEM:D7 treatment group compared to the control group. There was a greater (P < 0.004) percentage of morphologically normal developing follicles in the α-MEM:D1 treatment group compared to the TCM-199:D1 treatment group. Overall, the percentage of morphologically normal follicles was higher in the control group (72%; P < 0.02) and α-MEM:D1 group (84%; P < 0.0001) compared to the α-MEM:D7 (27%) treatment group. Mean follicle diameter was greater (P < 0.04) in the α-MEM:D7 treatment group (40.6 ± 1.1 μm) compared to the control group (37.3 ± 0.7 μm). Mean oocyte diameter was greater in the α-MEM:D1 (31.0 ± 0.7 μm; P < 0.006), TCM-199:D1 (30.7 ± 1.8 μm; P < 0.006), and α-MEM:D7 (33.2 ± 1.8 μm; P < 0.006) treatment groups compared to the control group (27.4 ± 0.9 μm). In conclusion, based on these preliminary data, in vitro culture of equine ovarian fragments obtained in vivo via the Biopsy Pick-Up method promoted preantral follicle development and follicle and oocyte growth in α-MEM for 7 days, with some follicles remaining morphologically normal throughout the culture period.

Effects of epidermal growth factor on in vitro maturation kinetics of porcine oocytes from different follicle classes

Effects of epidermal growth factor on in vitro maturation kinetics of porcine oocytes from different follicle classes

Study of Gene Expression in Bovine Granulosa Cells from Different Follicle Growth Phases.

It is known that 1 in 3 follicles contains a good a competent oocyte, but to this day the features of those particular follicles have not been characterized. The objective of this study was to build the transcriptomic profile of granulosa cells (GC's) originating from pre-dominance follicle (6 to 9mm diameter) in dairy cattle using microarrays and validating by Q-PCR. Granulosas originating from three different phases of antral follicle growth were compared: growing (G), plateau (P) and atresia (A), as categorized by flow cytometry on DNA content. The growing and atretic conditions we're each hybridized against the plateau condition as a reference in order to understand the specific biological mechanisms underway in this class of follicles, which is believed to hold oocytes with the best capacities to produce an offspring following in-vitro assisted reproduction technologies such as maturation (MIV) and fertilisation (FIV). 2,942 genes were differentially expressed (P<0.05) in G vs P and 1,974 in P vs A. Ingenuity Pathway Analysis (IPA) software was used to study the regulation pattern of genes and functions predicted to be enriched in the dataset. Cytometry was efficient in categorizing the follicles in the different phases, as determined by the down-regulation of steroidogenesis genes: cytochrome P450, subfamily XIX (CYP19A1, i.e. aromatase), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) and cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) in the granulosa cells of P follicles relative to G and probably in response to the decreasing FSH level. On the other hand, the P vs A contrast showed up-regulation of multiple transcripts associated to apoptosis: tissue transglutaminase (TGM2; transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferase)), SH3 domain containing ring finger 1(SH3RF1, also known as POSH), disabled-2 (DAB2), desmoglein-2 (DSG2), death receptor 5 (DR5), caspase-4 (CASP4), Ras association (RalGDS/AF-6) domain family member 1 (RASSF1), chaperonin containing TCP1, subunit 2 (beta) (CCT2) and interferon regulatory factor 5 (IRF5). Functions such as cell death, secretion of lipids and ploidy of cells, were predicted to be increased in P-follicle-granulosa compared to G-follicles, while functions such as growth of cells were predicted to be decreased in P relative to G. Although the genes more expressed in the A condition did not correspond to common atretic markers, they are likely to be indicators differentiating between the late atresia status of the follicle and early atresia, a hallmark characteristic of plateau follicles. This study offers multiple candidate genes to be further studied at a biochemical level in order to elucidate their role in the modulation of oocyte competence. This work was supported by NSERC of Canada.

IMMUNOHISTOCHEMICAL STUDY OF ESTROGEN AND PROGESTERONE RECEPTORS IN DIFFERENT SIZE CLASSES OF OVARIAN FOLLICLES IN DROMEDARY CAMELS

The present study aimed to investigate the normal distribution of estrogen (ERα) and progesterone (PR) receptors in the different classes of the ovarian follicles of the she-camel and their relation with the serum and follicular estrogen and progesterone hormonal level. Progesterone (P4) and 17β Estradiol (E2) serum levels, follicular estrogen receptors (Erα) and progesterone receptors (PR) expression and morphometry of different size classes of the ovarian follicles and corpus luteum during the estrous cycle in female dromedary camel were evaluated.The ERα and PR were detected using an indirect immunohistochemistry method (streptavidin-biotin immunoperoxidase method). Serum and follicular fluid were taken from 10 female dromedaries before and after slaughtering to evaluate the level of estrogen and progesterone in different size classes of the ovarian follicles (small >10mm, medium 10-15 and large <15 mm). Three cyclic non-lactating reproductively healthy she-camels were examined and bled daily to assess serum estrogen and progesterone levels during the follicular cycle and relate them to the size of the ovarian follicles and corpus luteum. The hormone levels were measured by radioimmunoassay. ERα was detected at low amounts in the follicular cells of the primordial, primary follicles and corpora lutea while, detected at moderate in the secondary follicles and high in the oocytes and in the tertiary follicles. On other hand, PR was detected in low reaction in secondary follicles and tertiary follicles. Moreover, it was estimated in a moderate reaction corpora lutea and corpora albicantia and stroma cells and in a strong reaction in the blood vessels. The number of cells expressing Erα and PR protein in ovarian cells was assessed by immunohistochemistry and computerized morphometric procedures. All data were analyzed with two way repeated measures ANOVA. Highest expression levels (p <0.01) of ER was found in the oolemma of the small and medium follicles but was absent in the granulosa cells surrounding these follicles. ERα was widely dispersed in the theca interna and theca externa of large tertiary follicles, it showed moderate staining intensity with homogeneous distribution in the cellular nucleus in theca interna and strong staining in the theca externa. The area and P4 serum concentration were higher during the luteal phase in R and C cows, as compared to F1. C and R animals displayed higher serum hormone levels than the F1 genotype, expression of mERα and glandular mPR and glandular sizes, which could pertain to the previously found better reproductive performance of C and R genotypes, probably as a result of their adaptation to a low-land tropical environmentExpression of PR was observed in stroma of all luteal structures but significantly stronger reaction was found in blood vessels of these structures. Estrogen concentration in both the follicular fluid (r=-0.06) and serum (r=-0.15) correlated negatively -but (not significantly)- with the size of the follicle while a positive non significant correlation was found between serum progesterone and the size of the corpus luteum. Serum and follicular fluid estrogen was higher in follicles exceeding 15 mm more than the lesser follicular categories. Slight difference in the concenttration of estrogen was found between follicles less than 10 mm in diameter and those between 10 – 15 mm. The expression of ER and PR and the secretion of their specific hormones in the ovary of she-camel were not always correlated with the presence of the hormones.

Ovarian abnormalities in a mouse model of fragile X primary ovarian insufficiency.

FMR1 premutation (PM) alleles have 55-200 CGG·CCG-repeats in their 5' UTR. PM carriers are at risk of fragile X-associated tremor and ataxia syndrome (FXTAS). Females are also at risk for FX primary ovarian insufficiency (FXPOI). PM pathology is generally attributed to deleterious properties of transcripts with long CGG-tracts. For FXPOI, hormone changes suggest a reduced residual follicle pool. Whether this is due to a smaller than normal original follicle pool or an increased rate of follicle depletion is unclear. A FX-PM mouse the authors generated with 130 CGG·CCG-repeats in the endogenous Fmr1 gene recapitulates features of FXTAS. Here the authors demonstrate that the gross development of the ovary and the establishment of the primordial follicle pool is normal in these mice. However, these animals show a faster loss of follicles of all follicle classes, suggesting that the problem is intrinsic to the ovary. In addition, many oocytes show aberrant nuclear accumulation of FMRP and elevated levels of ubiquitination. Furthermore, PM follicles are smaller and have fewer granulosa cells (GCs) than normal. Thus, these animals have ovarian abnormalities involving both the oocytes and GCs that may shed light on the molecular basis of FXPOI in humans.

Postpartum Ovarian Resumption in Native Dairy Cows in Upper Egypt and their Relation to Oxidant Antioxidant Status

The present study aimed to characterize the follicular pattern and ovarian resumption during the postpartum period in lactating dairy cows in Upper Egypt and study the impact of different components of the antioxidant system on follicular dynamics. Sixteen native dairy cows in their second lactation period were assigned for the current study. Cows were kept indoor and subjected to ultrasonographic examination and bled daily. Data regarding the change in the follicular dynamics and incidence of short cycles were collected and tabulated then statistically analyzed. Blood samples were used to determine nitric oxide (NO), Vitamin A, B-Carotene, Vitamin C and Vitamin E. results of the present study revealed that The first postpartum behavioral estrus observed 35-47 days (41.5 ± 1.86 days postpartum). The average postpartum days required for the animal to come in estrus in cows with transient CL was 44 ± 1.76 days and in cows without transient CL was 37 ± 1.18 days. In cows with transient corpus luteum, first ovulation was not accompanied by behavioral estrus (Quiet ovulation) at 23 ±1.02 days. Vit A, B-carotene, Vit E, NO, but not Vit C, increased significantly during the first observable postpartum heat in the studied cows and their level in the plasma correlated differently with the different classes of ovarian follicles. It is concluded that most antioxidants in the present study, except ascorbic acid, increases at the time of estrus and they are not affected by the day of postpartum period or the number and size of the dominant follicles. Short cycles are frequent in the postpartum cows (50% of cows had a short cycles in the present work). Follicular growth starts immediately – may be before the studied time- in postpartum dairy cows.

123 QUANTIFICATION, MORPHOLOGY AND VIABILITY OF EQUINE PREANTRAL FOLLICLES OBTAINED VIA BIOPSY PICKUP METHOD

To study early follicular development in the equine species, a biopsy pickup (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. The hypotheses tested in this study were that (1) preantral follicle quantity, viability and morphology do not differ according to stage of the oestrous cycle; (2) rate of atresia increases with follicle class; and (3) the BPU method provides sufficient material for studies on the early stages of folliculogenesis in mares. The transvaginal, ultrasound-guided BPU device was a 48-cm-long automated, spring-loaded device with an inner trocar point plunger containing a 15-mm specimen notch surrounded by an outer 16 G cutting needle (US Biopsy, Franklin, IN, USA). A total of 33 ovarian biopsy procedures were performed on 19 mares during the breeding season. Mares were 5 to 21 years of age and biopsies were performed during oestrus, diestrus, or both as confirmed by transrectal ultrasonography. Follicles were isolated mechanically using a tissue chopper and were analysed for either viability (live vs dead using trypan blue dye to analyse cell membrane integrity) or morphological normality (intact oocyte surrounded by granulosa cells that are well organised in 1 or more layers). Statistical analyses were performed using Fisher's exact test, paired t-test and one-way ANOVA. A total of 256 biopsy attempts were made, resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25 mg. An average of 18.8 ± 1.9 (mean ± s.e.m.) follicles were isolated per biopsy procedure. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). Primordial and primary follicles had an average diameter of 31.3 ± 6.2 μm and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P &lt; 0.001) for primordial follicles (91%) when compared with primary follicles (50%), with an overall follicle viability rate of 88%. Primordial follicles tended (P &lt; 0.06) to have a higher rate of morphological normality (96%) when compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested and number of follicles per milligram of tissue did not differ (P &gt; 0.05) according to reproductive status. Younger mares (ages 5 to 7) had more (P &lt; 0.05) follicles isolated per procedure than older mares (ages 14 to 21). The length of the interovulatory interval was not affected (P &gt; 0.05) by the biopsy procedure. No adverse effects were observed in cyclicity or general health following any procedure. The results supported the hypotheses tested. In conclusion, the BPU method provides material for the study of early follicular development in the equine species. In the future, the BPU method might be a feasible technique to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary to preserve genetic material or for large-scale embryo production.

Viability of oocytes and granulosa cells from cryopreserved ovine ovarian primordial, primary and secondary follicles

The study aimed to evaluate the effect of the addition of sucrose to the freezing and washing medium on the morphology and viability of ovine primordial, primary and secondary follicles and their enclosed oocytes and granulosa cells. Ovine primordial, primary and secondary ovarian follicles were cryopreserved in the absence or presence of 0.5M sucrose, with or without 1.0M ethylene glycol (EG) or 1.0M dimethyl sulphoxide (DMSO). After thawing, all follicles were washed in minimum essential medium (MEM), with or without 0.3M sucrose. Inclusion of sucrose in the freezing media generally improved the post-thaw morphology of the preantral follicles. The addition of sucrose to the washing media did not affect the percentage of normal follicles cryopreserved in the sucrose-containing media. However, its addition to the washing medium of ovarian tissue cryopreserved in the presence of EG, did increase the percentage of normal follicles. Although all the cryopreservation treatments lowered the percentage viability of the different isolated preantral follicle classes and their oocytes, the addition of sucrose to the cryoprotectants EG and DMSO appeared beneficial for the viability of all preantral follicles. The percentages recorded were lowest for the secondary follicles. In the presence of sucrose, cryopreservation of oocytes and granulosa cells from primordial and primary follicles was better than that from the cellular compartments of secondary follicles. It can thus be said that oocytes and granulosa cells of primordial and primary ovine follicles are well-cryopreserved in the presence of EG or DMSO, supplemented with sucrose, followed by a thawing-washing procedure in a sucrose-free medium.

Embryo development and follicular status of Toggenburg does fed urea diet

The effects of feeding urea on embryo production, quality and developmental stages and follicular status at the beginning of a superovulatory treatment of Toggenburg does fed urea diet were investigated in this study. Eighteen females were randomly allocated to receive diets with: no urea (control, n=8), and 2.4% urea dietary dry matter (n=10) UDM. The embryo recovery was performed at day 7 or 8 of estrus cycle by transcervical technique and classified according to quality and developmental stage. The follicular status was determined by transrectal ultrasonography at the first day of FSH injection. The number and quality of embryos were not affected by dietary urea concentration. Embryos from does treated with 2.4% UDM were recovered at advanced stage. Urea concentration (2.4%) decreases the number of follicles with less than 5 mm diameter. This follicle class is positively correlated to the number of recovered embryos and to the number and percentage of excellent and good embryos.

Detection of Follicular Apoptosis in Water Buffalo (Bubalus bubalis) Ovary by Histology and Nick End Labelling Technique

The objective of this experiment was to assess the features and extent of follicular apoptosis in the water buffalo (Bubalus bubalis) ovary using classical histology and nick end labelling technique. Ovaries (n=40) procured from the slaughterhouse were used for the study. The sections (5 μm) were used for detection of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) and classical histology (H&E). Those follicles showing ≥ 5% TUNEL positivity (TUNEL assay) and pyknotic nuclei (histology) in granulosa cells were classified as atretic. Based on histology, the atretic primary and secondary follicles (%) were 93.82 and 95.62 respectively. The histology study reveals that the rates (%) of atresia in <1, 1-3, 3-5 mm and >5 mm were 36.90, 40.50, 62.84 and 74.5 respectively. Further the atretic tertiary follicles (%) were significantly lower than the primary and secondary classes of follicles. TUNEL assay reveals that the atretic rate (%) of tertiary follicles in <1, 1-3, 3-5 and ≥ 5 mm class follicles were 50.88, 53.84, 81.81 and 36.36 respectively. The percentage of atresia in >5 mm diameter follicles were significantly lower in TUNEL than histology. Percentages of granulosa and thecal cells positive for atresia by TUNEL were 30.7 ± 0.53 and 13.82 ± 0.18 respectively per follicle. The initial structural changes in atretic follicles were seen primarily in the granulosa cells. In severely atretic follicles TUNEL positive granulosa cells along with theca cells have to be considered in assessing the rate and extent of atresia.

Are the levels of Cdk4 and Cx43 proteins of porcine oocytes associated with follicular size?

Abstract Gap junction connections are formed by proteins which play an important role in oocyte developmental competency but there is little information on the relationship between follicle size and the expression of genes encoding these proteins. The aim of this study was to investigate the potential association between follicle size and the levels of Cdk4 and Cx43 proteins using western blot analysis and confocal microscopic observations. Cumulus-oocyte complexes (COCs) were collected from puberal gilts (n = 20) of large (&gt;5 mm), medium (3-5 mm), and small (&lt;3 mm) follicles, and stained with BCB. BCB+ COCs, which had finished their growth phase, were cultured in TCM 199 for 44 h. Western blot analysis revealed an increased level of Cdk4 protein in oocytes isolated from large follicles as compared to medium (P &lt; 0.05) and small (P &lt; 0.01) ones. We did not detect differences in Cx43 protein levels in oocytes collected from any follicle class. Confocal microscopic observation revealed a specific membrane and zona pellucida localization of Cdk4 protein in oocytes isolated from large follicles, but an exclusively cytoplasmatic distribution of Cdk4 in oocytes from smaller follicle categories. The effect of follicular size on Cdk4 is indicated by the higher level of Cdk4 protein in oocytes isolated from large follicles and its variable distribution – perhaps resulting from a specific translocation mechanism – in the membrane, zona pellucida, and cytoplasm. IVM may also have a significant effect on Cdk4, as seen from the considerable difference in the expression and localization of Cdk4 protein in oocytes after IVM.

Ovarian Follicular Dynamics during the Estrous Cycle in Jennies in Upper Egypt

The objective of the current study was to describe follicular dynamics in Egyptian Jennies throughout the estrous cycle. In this experiment, 8 estrus cycles in 8 cyclic Jennies were studied from February to June using ultrasonography. The result revealed that one follicular wave per cycle was recorded throughout the studied period. Dominant follicle (DF) was firstly detected at −0.80 ± 0.84 day in Jennies. The growth rate of DF was 2.32 ± 0.18 mm/day. Left ovulations were nonsignificantly (P = .07) more than right ovulations (55.6% versus 44.6%). The CL was firstly detected at D 2.58 ± 1.2, developed in a rate of 1.19 ± 0.07 mm/day, reached a maximum diameter of 30.77 ± 1.28 mm at D 13.0 ± 0.70, and started to regress on D 17.05 ± 0.64 with a mean regression rate of 1.75 ± 0.17 mm d−1. Results of the present study indicated that Jennies had one follicular wave per cycle. The Day of the cycle has a significant effect on the number of different classes of the ovarian follicles, but not large ones. Ultrasonographic characteristics of the preovulatory follicles could be useful to predict ovulation. CL developed and regressed in a slow rate.

Regulation of folliculogenesis and the determination of ovulation rate in ruminants

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.