Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue

Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73-88%) than tissue frozen in 20% GLY or in either concentration of EG (16-52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80-97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either concentration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproductive cells within the ovary.     

Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk- and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 degrees C for 20 min in 1.8 ml of MEM containing 1.5 or 3M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3M GLY, as well as 3M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue.     

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.     

Immunocytochemical studies of relaxin in ovaries of pregnant and cycling mice

Immunocytochemical staining for relaxin in ovarian sections of pregnant mice from day 11 through day 18 of gestation revealed that only corpora lutea (CL) of pregnancy are stained. Evaluation of serial sections of ovaries from a day 16 pregnant mouse revealed that the only luteal structures present are CL of pregnancy. The number of CL present in each ovary equaled the number of implantation sites in each related horn (7 on the right side and 8 on the left side). These large CL varied in shape, being round in some profiles to very elongate in others. All CL were immunochemically stained for relaxin using the peroxidase-antiperoxidase method of L. Sternberger (Immunocytochemistry, 2nd ed. Wiley, New York, 1979). The intensity of the strain varied from cell to cell within each CL. Small luteal structures that were observed to be immunochemically stained for relaxin were demonstrated to represent the periphery of CL of pregnancy. No luteinized follicles were observed and interstitial cells and follicles were not immunochemically stained in any of the day 16 serial ovarian sections or in any of the ovarian sections from pregnant mice on the other days of gestation studied. CL of previous cycles were not observed to be present in the ovaries at days 15, 16, or 18 of gestation. However on day 14 and before, CL of previous cycles were observed and they did not exhibit any relaxin immunostaining. Immunocytochemical studies using the biotin-avidin system revealed that no relaxin immunostaining could be demonstrated in the ovaries of cycling mice at any stage of the estrous cycle. In conclusion, this study revealed that the only ovarian structures demonstrating relaxin immunocytochemical staining in the mouse were CL of pregnancy.     

Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

Background

The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep.

Methods

Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries).

Results

Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries.

Conclusion

These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.     

Depletion of ovarian follicles with age in chimpanzees: similarities to humans

We retrieved ovarian sections taken from necropsies of 19 captive chimpanzees (Pan troglodytes) aged 0-47 yr, counted the number of primordial follicles in each, and compared the rate of decline in numbers to declines previously documented in humans. The follicular depletion rate in this sample was indistinguishable from that shown across the same ages in classic human data sets. This result supports earlier suggestions that ovarian senescence occurs at the same ages in chimpanzees and humans, implying that the influence of declining ovarian function on other physiologic systems may be distinctively buffered in humans.     

Immunocytochemical localization and enzymatic distribution of rat ovarian aromatase

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.     

In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification

Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.     

Single-section counting error when distinguishing between primordial and early primary follicles in sections of rat ovary of different thickness

The number of primordial follicles within an ovary is frequently determined by counting 5, 7 or 10 microns thick sections and multiplying by the fraction of sections counted and a correction factor to adjust for duplicate counts. The objectives of the present study were: (i) to evaluate the accuracy of the correction factor developed by Abercrombie (1946); (ii) to evaluate the accuracy of the classification of primordial follicles from single tissue sections; and (iii) to determine the incorporation rate of 5-bromo-2-deoxyuridine into primordial follicles. In Expt 1, rat ovaries were sectioned at a thickness of 5, 7 or 10 microns. Primordial follicles were counted and classified across ten adjacent ovarian sections. The percentage of primordial follicles from single sections that were counted twice was 10, 9 and 2% in 5, 7 and 10 microns sections, respectively. This was lower than predicted by Abercrombie's method. The major error in counting from single sections was classification of early primary follicles as primordial follicles (55, 33 and 3% in 5, 7 and 10 microns sections, respectively). In Expt 2, a mean of 12 +/- 7% of primordial follicles incorporated 5-bromo-2-deoxyuridine after infusion for 7 days (four of seven rats had no labelled primordial follicles). In conclusion: (i) Abercrombie's correction factor should not be used for adjusting counts of follicles; (ii) evaluation of primordial follicles from single sections gives inaccurate counts and incorrect classification is of greater importance than duplicate counting, particularly in thinner sections; (iii) for evaluation of the number of follicles, 10 microns is the optimal thickness; and (iv) primordial follicles incorporated 5-bromo-2-deoxyuridine infrequently.     

Androgen receptor in early apoptotic follicles in the porcine ovary at pregnancy

Localization of androgen receptor (AR) was investigated in ovarian follicles developing and undergoing atresia during pregnancy in the pig. Immunohistochemical staining was conducted on ovarian antral follicles isolated on different days of gestation: 10, 18, 32, 50, 70, and 90. Paraffin sections were also subjected to in situ DNA labeling. TUNEL staining revealed the presence of positive follicles on all days of pregnancy but the amount of atretic follicles increased with time. However, even on day 90 of gestation many follicles were normal, with no signs of atresia. In atretic follicles, apoptotic cells were localized predominantly in the granulosa while theca was much less affected. Atretic follicles with many apoptotic cells were negative for AR. Nuclear immunostaining for AR was positive in follicles with limited amount of apoptotic cells. The same relationship was observed in ovarian follicles isolated at various days of pregnancy.     

In vitro binding of 125I-HCG to the rat ovary from birth to puberty

Summary Neither homogenates nor frozen ovarian sections of rats aged 1, 5 and 7 days demonstrated specific binding of ¹²⁵I-HCG. However, from day 10 to day 35, the homogenates could bind specifically, with an equilibrium association constant in the range of 0.9–2.7 × 10¹⁰ M^–1. The binding capacity increased from 5.8 fmol/mg tissue at day 10 to 15.7 fmol/mg tissue at day 35.As revealed by autoradiography, the frozen ovarian sections from 10-day-old rats showed ¹²⁵I-HCG binding localized over the interstitial and thecal tissues and, in the older age groups, also in the granulosa cells of follicles larger than 500 m in diameter.These results indicate that LH/HCG receptors appear in the rat ovary at the beginning of the second postnatal week, and that the interstitial cells are the main site of action of LH before puberty.     

Extra-ovarian production of mature viable mouse oocytes from frozen primary follicles

Isolated primary mouse follicles can be frozen successfully and thawed in the presence of 1.5 m-DMSO. Similar proportions of freshly collected and frozen-thawed primary follicles undergo folliculogenesis in the absence of other ovarian tissue. Some of the mature oocytes recovered from these follicles were fertilized in vitro and, after transfer to pseudopregnant recipients at the 2-cell stage, developed into live young. Cryopreservation and extra-ovarian development of immature follicles provide a unique opportunity to store large numbers of female gametes.     

Effect of the interval of serial sections of ovarian tissue in the tissue chopper on the number of isolated caprine preantral follicles.

The present work investigated the effect of the interval of serial sections of ovarian tissue on the number of isolated preantral follicles in the goat. Goat ovaries were cut in the tissue chopper at eight different intervals. The quality of isolated follicles were evaluated by histology and transmission electron microscopy. Best results were obtained when the ovaries were cut in the tissue chopper at intervals of 75.0 microm (9664 preantral follicles per ovary). Histochemical and ultrastructural analysis showed that the follicular morphology was preserved after mechanical isolation as demonstrated by the normality of oocytes and granulosa cells as well as by preservation of basement membrane. The percentages of isolated primordial, primary and secondary follicles were 96.3%, 2.5%, and 1.2% and their average diameters were 21.5, 34.7 and 65.3 microm, respectively. It was concluded that the interval of serial sections of ovarian tissue in the tissue chopper affects the number of isolated preantral follicles, and that the follicles remained intact after mechanical isolation in goats.     

Understanding follicle growth in vitro: Are we getting closer to obtaining mature oocytes from in vitro‐grown follicles in human?

Obtaining and fertilizing mature oocytes from immature follicles that were grown outside the body has conceptually attracted scientists for centuries, with initial attempts first documented in the 19th century. Significant progress has been made since then, due in part to a better understanding of folliculogenesis and improved techniques of in vitro follicle growth. Indeed, in vitro growth is now considered a reasonable approach to preserve or restore fertility when immature follicles and their oocytes need to be grown and matured outside the body. Certain patients would benefit from in vitro follicle growth, particularly those who carry a risk of cancer re‐seeding after grafting of frozen‐thawed ovarian tissue or who are at the risk of premature ovarian failure due to several intrinsic ovarian defects and genetic mutations that lead to accelerated follicle atresia and early exhaustion of the ovarian reserve. This review provides an update on the current status of in vitro growth of preantral human follicles, from initial efforts to the most recent achievements.     

Histological and ultrastructural analysis of cryopreserved sheep preantral follicles

The aim of this study was to verify the histological and ultrastructural characteristics of sheep preantral follicles after exposure of ovarian tissue to cryopreservation in glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) in order to determine the optimum method to store sheep ovarian tissue for later experimental or clinical use. Each ovarian pair from five mixed-breed ewes was divided into 17 fragments. One (control) fragment was immediately fixed for routine histological and ultrastructural studies and the remaining (test) fragments were randomly distributed in cryotubes, equilibrated at 20 degrees C/20 min in 1.8 mL of minimal essential medium (MEM) containing 1.5 or 3 M GLY, EG, PROH or DMSO and then either fixed for morphological studies to determine their possible toxic effect or frozen/thawed and then fixed to test the effect of cryopreservation on preantral follicles. Histological analysis showed that, compared to control fragments, all cryoprotectants at both concentrations significantly reduced the percentage of normal preantral follicles in ovarian fragments prior to or after cryopreservation. PROH 3.0 M appeared to exert a more toxic effect (P<0.05) than the other cryoprotectants in noncryopreserved tissues. After freezing/thawing, the highest (P<0.05) percentages of lightmicroscopical normal preantral follicles were observed in ovarian fragments cryopreserved in EG (1.5 and 3 M) or DMSO (1.5 M). However, transmission electronic microscopical (TEM) examination showed that only the DMSO-cryopreserved preantral follicles had normal ultrastructure. The data suggest that sheep preantral follicles should be cryopreserved with 1.5 M DMSO for later clinical or experimental application.     

Development of in vitro culture method for early stage zebrafish (Danio rerio) ovarian follicles for use in cryopreservation studies

There have been no reported methods for in vitro growth of early stage ovarian follicles for fish and their cryopreservation is still under investigation. If cryopreservation of early stage ovarian follicles can be achieved, in vitro procedures for ovarian follicle culture, development, ovulation and fertilisation after cryopreservation would be needed. The aim of the present study was to develop an in vitro culture method for early stage zebrafish ovarian follicles for use after their cryopreservation. Procedures for in vitro culture of stage I (primary growth) and stage II (cortical alveolus) ovarian follicles were developed. The effects of concentration of L-15 medium, pH and the concentration of human chorionic gonadotropin (hCG) and activin A were studied. The results demonstrated that early stage zebrafish ovarian follicles can be cultured in vitro for 24 h, stage I and II ovarian follicles can grow to the sizes of early stage II and stage III ovarian follicles after hCG treatment. The method developed here is effective for assessing early stage zebrafish ovarian follicles growth competence in vitro. The results from the present study indicated that in vitro culture is the most reliable method for assessing ovarian follicle viability when compared with vital dye staining methods.     

Altered expression of Bcl-2 and Bax in follicles within dehydroepiandrosterone-induced polycystic ovaries in rats

PCOS (polycystic ovary syndrome) is a heterogeneous disease characterized by hyperandrogenaemia, hirsutism, oligo- or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA (dehydroepiandrosterone) administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of AFs (antral follicles) in both groups. Bax expression was greater in preantral and AFs from PCOS ovarian sections than in the controls. In contrast, intense Bcl-2 immunostaining was observed in the control AFs, while Bcl-2 protein was either absent in PFs (preantral follicles) or weakly expressed in AFs from PCOS rats. These results were partially confirmed by Western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in the control and that there were no differences in Bax ovarian levels between groups. However, Bax/Bcl-2 ratio was significantly higher in PCOS group than in the control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEA-induced PCOS rats.     

Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections,imprint cytology and immunocytochemistry

M. Francz, K. Egervari and Z. Szollosi
Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections, imprint cytology and immunocytochemistry Objective: We analysed the utility of imprint cytology with rapid immunocytochemistry and frozen section analysis for the evaluation of sentinel lymph nodes in breast cancer patients. Methods: The sensitivity, specificity, and positive and negative predictive values have been calculated for each method individually, each pair and all three together. We compared these results with those of routinely processed paraffin sections. Results: The sensitivity and specificity of each of the three methods for detection of metastatic carcinoma were as follows: 69.4% and 97.8% for touch imprint cytology; 58.3% and 100% for frozen sections; 68.5% and 98.9% for rapid immunocytochemistry. When the methods were combined, the highest accuracy was achieved by touch imprint cytology, frozen sections, touch imprint cytology plus rapid immunocytochemistry, or touch imprint cytology frozen section analysis and rapid immunocytochemistry, each of these having identical sensitivity and specificity of 72.2% and 97.8%, respectively. Conclusions: In our study the combined accuracy of the three methods was the same as combining touch imprint cytology and frozen sections or touch imprint cytology plus rapid immunocytochemistry. Rapid immunocytochemistry provides an additional parameter and preserves tissue for permanent sections.     

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-α and cytochrome P450aromatase genes in the rat ovary

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-α subunit and cytochrome P450 aromatase (P450_arom) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100–200 μm maximum diameter) and ten healthy antral ( > 300 μm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-α, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-α subunit and P450_arom were quantified by real-time polymerase chain reaction (PCR). While the amount of P450_arom mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-α mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-α subunit mRNA independently of antral formation in immature rat ovaries.     

Circulating gonadotropins levels and contribution of different large antral follicles to isofolliculia in sheep

The aim of this study was first to search for isofolliculia in right and left ovaries during postnatal development of Ouled Djellel ewe lambs, a non-seasonal breed of sheep. In addition, the contribution of different sizes of large antral follicles to this phenomenon was studied, and finally the variations in both plasma FSH and LH levels during this period of life were determined. Plasma was collected from groups of four ewe lambs at 0 (<24 h), 1 week, and every 2 weeks from 4 to 14 weeks of age. Thereafter, each group was slaughtered, right and left ovaries recovered, weighed and their length and width measured. One ovary was fixed in Bouin–Holland's solution and prepared for histological study. The other one was immediately frozen and cut in a cryostat and prepared for histochemical study. This latter method was used to detect the activity of alkaline phosphatase, lactate dehydrogenase, NADH₂-tetrasolium reductase, and glucose-6-phosphate dehydrogenase enzymes. Number and size of antral follicles and their contribution to isofolliculia were determined from ovarian sections of both studies. Isofolliculia was seen in right and left ovaries of Ouled Djellel ewe lambs at 4, 6, 8, and 10 weeks of age. This phenomenon was characterized by the presence of large antral follicles almost equal in size and total enzymatic inactivity in the interstitium. Weight and dimensions of right and left ovaries increased rapidly from birth to 4 weeks of age, and then rose gradually to week 8 and then rising again to week 10, followed by a decline at 12 and 14 weeks of age. All large antral follicles contributed to isofolliculia in right and left ovaries but, the percentage of antral follicles <2 mm at 4, 6, 8, and 10 weeks of age were significantly greater than the percentage of follicles ≥2 and <3 mm and the contribution of follicles ≥3 mm was the lowest. FSH levels increased slowly from birth to 6 weeks of age then, increased rapidly to week 10, followed by a decline at weeks 12 and 14. LH was low at birth and the level increased slowly to 8 weeks of age, followed by a further rapid increase at 10 weeks of age. All parameters studied did not show any significant differences between the right and left ovary. It was concluded that isofolliculia occurred between 4 and 10 weeks of age in left and right ovaries of Ouled Djellel ewe lambs. This phenomenon was characterized by the increase of both ovarian weights and dimensions, and of plasma FSH and LH levels. All large antral follicles ≥1 mm in diameter contributed to isofolliculia but the contribution of antral follicles <2 mm was greater than the contribution of antral follicles ≥2.