Mature Rat Ovary Research Articles

The decellularized ovary as a potential scaffold for maturation of preantral ovarian follicles of prepubertal mice

ABSTRACT In the current study, a decellularized ovary scaffold was used for the encapsulation and development of preantral follicles. Maturation and the expression of the related genes were evaluated and compared against in vivo conditions. Mature rat ovaries were decellularized and decellularization was confirmed using hematoxylin/eosin and Hoechst stains. Collagen and glycosaminoglycan (GAG) preservation and the extracellular matrix (ECM) architecture were evaluated by Masson’s trichrome staining, Alcian blue staining, and scanning electron microscopy, respectively. Raman confocal microscopy and DNA quantification were performed to show DNA and cell debris depletion and ECM retention. Preantral follicles of mice were cultured for 12 days in the decellularized (3D) and two-dimensional (2D) conditions. Finally, the survival rate, antrum formation rate and diameters of follicles, levels of estradiol (E2) and progesterone (P4), and expression of the ZP2, Gdf9, Bmp6, and Bmp15 genes were evaluated in the 2D and 3D culture systems. On day 13 of culture, oocyte maturation was induced by hCG. Although histochemical tests showed partial retention of ECM, Raman confocal microscopy showed some extent of ECM washing out along with the depletion of DNA and cell debris. However, SEM showed the preservation of the ovarian ECM’s ultrastructure. The survival rate of follicles was similar, but the antrum formation rate, concentrations of E2 and P4, and oocyte maturation rate were significantly higher in 3D compared with 2D. The level of ZP2, Gdf9, Bmp6, and Bmp15 gene expression in follicles matured in 2D, 3D, and in vivo was statistically similar. In all matured follicles, their gene expression levels significantly declined relative to preantral follicles. Ovarian follicles that matured in the decellularized ovaries demonstrated the same follicular growth and maturation gene expression levels as those that grew in the natural ovary. This is consistent with using the decellularized ovary as a scaffold suitable for ovarian reconstruction. Abbreviations GAG: glycosaminoglycan; ECM: extracellular matrix; 2D: two-dimensional; E2: estradiol; P4: progesterone; BMP15: bone morphogenetic protein 15; GDF9: growth differentiation factor 9; ZP2: zona pellucida 2; Gdf9: growth/differentiation factor-9; Bmp6: bone morphogenetic protein 6; Bmp15: bone morphogenetic protein 15.

MORPHOFUNCTIONAL PROPERTIES OF THE RATS BLOOD CELLS IN THE DEVELOPMENT OF EXPERIMENTALLY INDUCED ESTROGEN-DEPENDENT OVARIAN TUMORS

Using the model of the experimental hormone-dependent tumor of the ovaries of mature female rats, the morphofunctional properties of the blood constituents closely associated with neoplastic ovarian cells were studied. Using modern technologies of AFM scanning and confocal laser scanning microscopy, it is shown that the development of the tumor process in the body is accompanied by a decrease in viability and an increase in the number of dead lymphocytes. Functionally, the leading populations that respond to the development of the tumor process in the body are leukocytes, which are characterized by the reactions of spreading and smoothing out the microrelief of the surface. The development of the tumor process is accompanied by a change in the elasticity of blood cells, which allows early diagnosis of tumor transformations in various tissues and organs.

Apoptosis in autotransplanted cryopreserved rat ovarian tissues

OBJECTIVE: To evaluate apoptosis in autoransplanted rat ovarian tissues after Vitrification (V) or Slow Freezing (SF) cryopreservation. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Mature rat (n=14) ovaries were placed in phosphate buffered saline (PBS) and randomly assigned to one of two treatments: V plus autotransplantation (VA) or SF plus autotransplantion (SFA). The V (n=8) was done using 1M dimethylsulfoxide(DMSO) followed by 2M DMSO + 1M Acetamide + 1M propylene glycol in PBS. Ovaries were thawed at 25°C for 30 sec in PBS containing 0.25M sucrose. SF (n=6) was done in 1.5 M DMSO and 0.1 M Sucrose. Ovaries were initially cooled to -6°C at -2°C/min, ice nucleated, cooled at -0.3°C/min to -40°C then at -10°C/min to -80°C then plunged in LN. Thawing was done at 25°C using 0.75M DMSO, and 0.25M sucrose. Ovarian autotransplantation (n=5) was performed within 1 h of thawing. Pre-freeze, post-freeze and post-transplant sections were microscopically analyzed for apoptosis with DNA staining using DAPI and TUNEL. Results were analyzed by ANOVA with Tukey post-hoc testing and paired- t-test. RESULTS: Follicular apoptosis was lower in pre-frozen ovarian tissue as compared to post-frozen tissue in SF and V methods (p=0.0478 and p=0.0009 respectively). No difference was found in the number of total follicles between SF and V methods but the amount of damage to living follicular cells was increased in V (p=0.0071). After transplantation, the number of apoptotic cells was lower in pre-frozen tissue than in post-transplanted tissue (p=0.0001). No significant difference was seen in the amount of apoptotic cells between post-frozen and post-transplanted ovarian tissue, suggesting that more damage to the follicles occurred during the freezing/thawing process than by autotransplantation. CONCLUSIONS: As indicated by apoptosis, SF may be less traumatic, post thaw, than V and the freezing/thawing process contributes more to the follicular loss than autotransplantation.

Gene expression profile of rat ovarian tissue following xenotransplantation into immune-deficient mice

Immune-compromised mice have been used as gonadal tissue recipients to develop gametes of various mammalian species. The aim of this research was to determine gene expression differences between fresh and frozen-thawed rat xenotransplanted (XT) ovaries as well the gene expression differences between XT and sexually mature rat ovaries that were non-transplanted (NT). Ovaries from sexually immature female rats were transplanted under the kidney capsule of ovariectomized athymic nude mice either fresh or after freezing. The XT ovaries were collected approximately 10-12 weeks after xenografting for microarray analysis. The NT ovaries were collected from sexually mature rats. Gene expression was very similar between fresh and cryopreserved XT ovaries: 125 genes were twofold up- or downregulated, but level of regulation was not statistically significant. Overall patterns of gene expression between XT and NT ovaries were very different indicated by the absence of diagonal relationship between XT and NT ovary gene expression. More than 3000 genes were significantly (P<0.01) up- or downregulated between XT and NT ovaries. Genes involved in metabolic processes, lipid metabolism, and growth were downregulated in XT ovaries, whereas genes involved in immune and inflammatory response were upregulated in XT ovaries. The results showed that ovarian tissue xenografting significantly alters genes responsible for ovarian metabolism and function and leads to an upregulation of genes responsible for graft rejection.

HTRA3 expression in non-pregnant rhesus monkey ovary and endometrium, and at the maternal-fetal interface during early pregnancy

BackgroundHTRA3 is a recently identified member of the mammalian serine protease family HTRA (high temperature requirement factor A). In both the rodent and the human HTRA3 is transcribed into two mRNA species (long and short) through alternative splicing. We have previously shown that HTRA3 is expressed in the mature rat ovary and may be involved in folliculogenesis and luteinisation. HTRA3 is also upregulated during mouse and human placental development. The current study investigated whether HTRA3 is also localised in the primate ovary (rhesus monkey n = 7). In addition, we examined the non-pregnant rhesus monkey endometrium (n = 4) and maternal-fetal interface during early pregnancy (n = 5) to further investigate expression of HTRA3 in primate endometrium and placentation.MethodsHTRA3 mRNA levels in several rhesus monkey tissues was determined by semiquantitative RT-PCR. Protein expression and localisation of HTRA3 was determined by immunohistochemistry.ResultsLong and short forms of HTRA3 mRNA were detected in the ovary, aorta, bladder, small intestine, skeletal muscle, heart and uterus but not the liver nor the kidney. HTRA3 protein was immunolocalised to the oocyte of all follicular stages in the rhesus monkey ovary. Protein expression in mural and cumulus granulosa cells of late secondary follicles increased significantly compared to granulosa cells of primordial, primary and secondary follicles. Mural and cumulus granulosa cells of antral follicles also showed a significant increase in expression. Staining intensity was higher in the granulosa-lutein cells compared to the theca-lutein cells of corpora lutea (n = 3). In the non-pregnant monkey endometrium, HTRA3 was detected in the glandular epithelium. The basalis endometrial glands showed higher staining intensity than functionalis endometrial glands. During early pregnancy, strong staining for HTRA3 protein was seen in both maternal decidual cells and glands.ConclusionWe propose that HTRA3 may be involved in folliculogenesis and luteinisation in the primate ovary. Furthermore, similar to previous findings in the human, HTRA3 is possibly a factor involved in and potentially important for primate placentation.

Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary.

Leptin is secreted by adipocytes and exerts its effects by interacting with the long form of the leptin receptor, OB-RB. The leptin protein and leptin receptors have been localized in the ovary, and acute leptin treatment directly inhibits ovulation in the rat ovary. It was hypothesized that expression of the leptin receptor gene varies throughout the oestrous cycle to modulate the sensitivity of the ovary to leptin. In this study, expression of genes for the long and short isoforms of the leptin receptor in the adult ovary was investigated at different stages of the rat oestrous cycle. Vaginal cytology was used to determine the stage of the oestrous cycle. Ovaries were collected and RNA was extracted for real-time RT-PCR analysis of leptin receptor gene expression. OB-RB gene expression was low in pro-oestrus (3.13 +/- 0.18 fg RNA per microg total DNA) and dioestrus II (2.52 +/- 0.19 fg RNA per microg total DNA) of the oestrous cycle, whereas expression was high in oestrus (5.9 +/- 0.27 fg RNA per microg total DNA) and dioestrus I (4.6 +/- 0.24 fg RNA per microg total DNA) (P < 0.001). Expression of the gene for the short form of the leptin receptor (OB-RA) was at a maximum in dioestrus I (65.5 +/- 0.8 fg RNA per ng total DNA), high in oestrus (39.0 +/- 0.8 fg RNA per ng total DNA) and low at pro-oestrus (5.0 +/- 0.2 fg RNA per ng total DNA) and dioestrus II (1.1 +/- 0.09 fg RNA per ng total DNA) (P < 0.001). Plasma oestradiol concentrations (pg ml-1) were highest at pro-oestrus (19.38 +/- 1.3), and similar at the remaining three stages studied (oestrus: 13.7 +/- 1.9; dioestrus I: 12.4 +/- 1.0; dioestrus II: 10.3 +/- 0.9) (P < 0.05). Plasma progesterone concentrations (ng ml-1) were higher in the luteal phases of the oestrous cycle (dioestrus I: 18.6 +/- 2.3; dioestrus II: 14.7 +/- 2.5) than during pro-oestrus (5.12 +/- 0.6) and oestrus (5.9 +/- 0.8) (P < 0.05). Plasma leptin concentrations were detectable only in pro-oestrus (0.35 +/- 0.05 ng ml(-1)) and were below the detection limit of the assay at other stages of the oestrous cycle. In summary, mRNA content for the long and short isoforms of the leptin receptor is lower in pro-oestrus and dioestrus II than in oestrus and dioestrus I of the rat oestrous cycle. The fluctuations in leptin receptor mRNA content may be a response to the concentrations of circulating steroid hormones and leptin. This research supports the initial hypothesis and shows that ovarian leptin receptor concentrations vary throughout the oestrous cycle in response to the changing environment of the ovary.

Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary

Leptin is secreted by adipocytes and exerts its effects by interacting with the long form of the leptin receptor, OB-RB. The leptin protein and leptin receptors have been localized in the ovary, and acute leptin treatment directly inhibits ovulation in the rat ovary. It was hypothesized that expression of the leptin receptor gene varies throughout the oestrous cycle to modulate the sensitivity of the ovary to leptin. In this study, expression of genes for the long and short isoforms of the leptin receptor in the adult ovary was investigated at different stages of the rat oestrous cycle. Vaginal cytology was used to determine the stage of the oestrous cycle. Ovaries were collected and RNA was extracted for real-time RT-PCR analysis of leptin receptor gene expression. OB-RB gene expression was low in pro-oestrus (3.13 +/- 0.18 fg RNA per microg total DNA) and dioestrus II (2.52 +/- 0.19 fg RNA per microg total DNA) of the oestrous cycle, whereas expression was high in oestrus (5.9 +/- 0.27 fg RNA per microg total DNA) and dioestrus I (4.6 +/- 0.24 fg RNA per microg total DNA) (P < 0.001). Expression of the gene for the short form of the leptin receptor (OB-RA) was at a maximum in dioestrus I (65.5 +/- 0.8 fg RNA per ng total DNA), high in oestrus (39.0 +/- 0.8 fg RNA per ng total DNA) and low at pro-oestrus (5.0 +/- 0.2 fg RNA per ng total DNA) and dioestrus II (1.1 +/- 0.09 fg RNA per ng total DNA) (P < 0.001). Plasma oestradiol concentrations (pg ml-1) were highest at pro-oestrus (19.38 +/- 1.3), and similar at the remaining three stages studied (oestrus: 13.7 +/- 1.9; dioestrus I: 12.4 +/- 1.0; dioestrus II: 10.3 +/- 0.9) (P < 0.05). Plasma progesterone concentrations (ng ml-1) were higher in the luteal phases of the oestrous cycle (dioestrus I: 18.6 +/- 2.3; dioestrus II: 14.7 +/- 2.5) than during pro-oestrus (5.12 +/- 0.6) and oestrus (5.9 +/- 0.8) (P < 0.05). Plasma leptin concentrations were detectable only in pro-oestrus (0.35 +/- 0.05 ng ml(-1)) and were below the detection limit of the assay at other stages of the oestrous cycle. In summary, mRNA content for the long and short isoforms of the leptin receptor is lower in pro-oestrus and dioestrus II than in oestrus and dioestrus I of the rat oestrous cycle. The fluctuations in leptin receptor mRNA content may be a response to the concentrations of circulating steroid hormones and leptin. This research supports the initial hypothesis and shows that ovarian leptin receptor concentrations vary throughout the oestrous cycle in response to the changing environment of the ovary.

Gonadotropin-dependent expression of sterol 14-demethylase P450 (CYP51) in rat ovaries and its contribution to the production of a meiosis-activating steroid.

Immunohistochemical analysis showed that sterol 14-demethylase P450 (CYP51) is expressed in mature follicles and corpus lutea of rat ovaries. In follicles, CYP51 is expressed in granulosa and theca cells but not in oocytes. The ovarian CYP51 activity of hypophysectomized rats is very low and induced by pregnant mares' serum gonadotropin (PMSG) treatment together with ovarian growth. The expression of CYP51 first increases in growing follicles and then appears in the corpus lutea after luteinization. The former event may be due to the follicular-stimulating hormone action of PMSG, and the latter may be caused by the luteinizing hormone effect of PMSG. Sterol analysis indicated that the product of the CYP51-mediated lanosterol 14-demethylation, 4,4-dimethylcholesta-8,14,24-trienol, which has been identified as a meiosis-activating steroid (MAS) in mammals [Byskov et al. (1995) Nature 374, 559-562], accumulates (about 10 pmol/mg of ovary) in mature rat ovaries, and the content is enough to activate the resumption of meiosis. These lines of evidence suggest that the expression of ovarian CYP51 is dependent on gonadotropins, and ovarian CYP51 activity is enough for accumulating MAS. Serum insulin does not affect the ovarian CYP51 level, although it is essential for hepatic CYP51 expression. These findings indicate that expression of CYP51 is regulated differently among organs.

Comparison of Nuclear Magnetic Resonance Microimages and Histology of Rat Ovaries.

Nuclear magnetic resonance (NMR) microscopy is a magnetic resonance imaging technique with an enhanced spatial resolution. In this study, fresh ovaries of mature rats immersed in phosphate buffered saline (PBS) were examined by NMR microscopy. Histological sections corresponding to NMR microimages were prepared, and components in ovaries were identified in microimages by comparison with light microscopic images. Signal intensities of antral follicles, corpora lutea (CL), stroma, adjacent fat tissue and PBS were measured in images with different echo times (TE) and analyzed. As TE increased, signal intensities decreased in CL and connective tissue including the stroma, while those in PBS and follicles changed less. Fat showed significantly high signals compared with other components at each TE. Stroma showed significantly low signals than other components except PBS and follicles at TE of 5 msec. There were no significant differences among CL, follicles and PBS at TE of 5 msec. At TE of 20 msec, however, there were significant differences in signal intensities between each components in the rank order fat > PBS > follicles > CL > stroma. Significant regression of signal intensity was seen on TE in follicles, CL and stroma. NMR microscopy is considered to be a useful tool to investigate the physiological function of rat ovaries.

P-243. The cell-specific expression of the angiotensin II-receptor type 2 (AT2) and its functional significance in the mature rat ovary

P-243. The cell-specific expression of the angiotensin II-receptor type 2 (AT2) and its functional significance in the mature rat ovary

Steroidogenic factor-1 regulates the rate of proliferation of normal and neoplastic rat ovarian surface epithelial cells in vitro.

Steroidogenic factor-1 (SF-1) is a transcription factor that is expressed by many cell types within the ovary and has been shown to inhibit granulosa cell proliferation. The present studies were designed to determine whether: 1) SF-1 is expressed by primary and transformed rat ovarian surface epithelial cells (i.e. ROSE cells); and 2) SF-1 expression effects the proliferation of both normal and neoplastic ROSE cells. These studies used immature, gonadotropin-primed and mature rat ovaries, as well as ROSE-179 cells from early passages (EP) and late passages (LP), T-sv-40 transformed ROSE cells, and T-ras transformed ROSE cells. In situ hybridization studies demonstrated that SF-1 was detected in the surface epithelium of rat ovaries, independent of age or gonadotropin treatment. Further, Northern blot and quantitative in situ hybridization studies revealed that significant amounts of SF-1 messenger RNA (mRNA) were present in EP-ROSE-179 cells but not in the other cell lines. Interestingly, EP-ROSE-179 cells proliferated at a significantly slower rate than the other cell lines. Further, SF-1 mRNA levels were higher in EP-ROSE-179 cells in the G0/G1 stage than in the S-, G2/M stage of the cell cycle. These observations suggest that a cause and effect relationship exists between the level of SF-1 expression and cell proliferation. To test this hypothesis, LP, T-sv-40, and T-ras ROSE cells were transfected with either control vector or SF-1 expression vector. Forty-eight hours after transfection, SF-1 expression was assessed by in situ hybridization, and the fold increase in cell number/24 h was determined. For each cell line, about 30% of the cells were successfully transfected. The fold increase in the number of cells observed after transfection with the SF-1 expression vector was significantly less than the increase in cell number after transfection with the control vector (P < 0.05). To confirm that the forced expression of SF-1 prevented proliferation, LP cells were cotransfected with a green fluorescent protein (GFP) expression vector and either control vector or SF-1 expression vector. This study demonstrated that virtually none of the GFP/SF-1-transfected cells proliferated over a 24-h period, whereas GFP/Control vector-transfected cells proliferated. Further, approximately 40% of the GFP/SF-1-transfected cells underwent apoptosis after 24 h of culture in serum-supplemented medium. These data demonstrate that: 1) normal ovarian surface epithelial cells express SF-1; 2) SF-1 is also expressed by EP-ROSE-179 cells, but its expression seems to be suppressed when the cells enter the cell cycle; 3) LP-, T-sv, and T-ras ROSE cells do not express SF-1 mRNA; and 4) the inability to express SF-1 is associated with an increase in cell proliferation. Finally, forced SF-1 expression interferes with serum-induced proliferation and leads to apoptosis.

Steroidogenic Factor-1 Regulates the Rate of Proliferation of Normal and Neoplastic Rat Ovarian Surface Epithelial Cells in Vitro

Steroidogenic factor-1 (SF-1) is a transcription factor that is expressed by many cell types within the ovary and has been shown to inhibit granulosa cell proliferation. The present studies were designed to determine whether: 1) SF-1 is expressed by primary and transformed rat ovarian surface epithelial cells (i.e. ROSE cells); and 2) SF-1 expression effects the proliferation of both normal and neoplastic ROSE cells. These studies used immature, gonadotropin-primed and mature rat ovaries, as well as ROSE-179 cells from early passages (EP) and late passages (LP), T-sv-40 transformed ROSE cells, and T-ras transformed ROSE cells. In situ hybridization studies demonstrated that SF-1 was detected in the surface epithelium of rat ovaries, independent of age or gonadotropin treatment. Further, Northern blot and quantitative in situ hybridization studies revealed that significant amounts of SF-1 messenger RNA (mRNA) were present in EP-ROSE-179 cells but not in the other cell lines. Interestingly, EP-ROSE-179 cells proliferated at a significantly slower rate than the other cell lines. Further, SF-1 mRNA levels were higher in EP-ROSE-179 cells in the G0/G1 stage than in the S-, G2/M stage of the cell cycle. These observations suggest that a cause and effect relationship exists between the level of SF-1 expression and cell proliferation. To test this hypothesis, LP, T-sv-40, and T-ras ROSE cells were transfected with either control vector or SF-1 expression vector. Forty-eight hours after transfection, SF-1 expression was assessed by in situ hybridization, and the fold increase in cell number/24 h was determined. For each cell line, about 30% of the cells were successfully transfected. The fold increase in the number of cells observed after transfection with the SF-1 expression vector was significantly less than the increase in cell number after transfection with the control vector (P < 0.05). To confirm that the forced expression of SF-1 prevented proliferation, LP cells were cotransfected with a green fluorescent protein (GFP) expression vector and either control vector or SF-1 expression vector. This study demonstrated that virtually none of the GFP/SF-1-transfected cells proliferated over a 24-h period, whereas GFP/Control vector-transfected cells proliferated. Further, approximately 40% of the GFP/SF-1-transfected cells underwent apoptosis after 24 h of culture in serum-supplemented medium. These data demonstrate that: 1) normal ovarian surface epithelial cells express SF-1; 2) SF-1 is also expressed by EP-ROSE-179 cells, but its expression seems to be suppressed when the cells enter the cell cycle; 3) LP-, T-sv, and T-ras ROSE cells do not express SF-1 mRNA; and 4) the inability to express SF-1 is associated with an increase in cell proliferation. Finally, forced SF-1 expression interferes with serum-induced proliferation and leads to apoptosis.

Granulosa cell DNA synthesis is strictly correlated with the presence of insulin-like growth factor I and absence of c-fos/c-jun expression.

Insulin-like growth factor I (IGF-I) mRNA is selectively expressed by a subset of rat ovarian follicles. To elucidate the biological role and regulation of follicular IGF-I, we have analyzed individual follicles with respect to IGF-I production, DNA synthesis, and c-fos and c-jun expression, since AP-1 complex components have been implicated both in regulation of IGF-I gene expression and cell proliferation. IGF-I mRNA localization, bromodeoxyuridine (BRDU) incorporation, and c-fos and c-jun immunoreactivity were compared in serial sections from prepubertal and mature rat ovaries. BRDU incorporation is exclusively concentrated in IGF-I-expressing follicles in both prepubertal and mature rats, with further intrafollicular correlation between local concentrations of BRDU-positive granulosa cells and IGF-I mRNA. Furthermore, there is a striking negative correlation between IGF-I mRNA localization and follicular fos/jun immunoreactivity. Both c-fos and c-jun are readily detected in granulosa cells of luteinized follicles where IGF-I mRNA is never found. Immunoreactive c-jun but not c-fos is detected in granulosa cells of atretic follicles, while neither c-fos nor c-jun is detected in IGF-I mRNA-positive granulosa cells. These data implicate IGF-I in granulosa cell proliferation and the fos/jun heterodimer in granulosa cell luteinization, while expression of c-jun in the absence of c-fos may be implicated in programmed granulosa cell death.

Tokishakuyakusan Effect on DNA Polymerase α Activity in Relationship to DNA Synthesis before and/or after the LH/FSH Surge in Rats

DNA polymerase alpha activity in ovaries of mature cycling rats during the normal estrous cycle changed in a cyclic manner with a peak at 1800 h in proestrus. Tokishakuyakusan (TS) in vivo did not affect the changes in DNA polymerase alpha and beta activities during the estrous cycle. LH and FSH at 1000 or 1700 h in proestrus increased DNA polymerase alpha activity, but the DNA polymerase alpha activity induced by LH or FSH was not significantly affected by the addition of TS. DNA polymerase beta activity did not change with LH, FSH or TS. In PMS-treated or -untreated immature rats, TS enhanced ovarian DNA polymerase alpha activity but had no significant effect on LH or FSH action. In ovaries, incubated in vitro, in untreated mature or immature rats, TS enhanced ovarian DNA polymerase alpha activity but had no significant effect on LH or FSH action. These results suggest that TS stimulates ovarian DNA polymerase alpha activity in relationship to DNA synthesis and does not affect the effect of LH or FSH on the activity by preovulatory follicle before and/or after the LH/FSH surge.

A Novel 34-kDa Glutathione-Binding Protein in Mature Rat Ovary

A novel protein that binds to a glutathione-Sepharose affinity column has been detected in mature, but not immature,rat ovary. This protein could be resolved from all identifiable components when the affinity-purified material, containing primarily glutathione transferases, was analyzed on reversed phase-HPLC. The unidentified protein migrated with an apparent molecular weight of 34 kDa on SDS-PAGE. After purification by affinity chromatography and subsequent preparative electrophoresis, the protein was subjected to N-terminal amino acid sequence analysis. The sequence obtained demonstrated a high degree of homology with an internal amino acid sequence in human carbonyl reductase (EC 1.1.1.184).

Immunoelectron microscopic localization of laminin in rat ovarian follicles.

We studied the immunohistochemical and ultrastructural distribution of laminin in ovaries of immature and mature rats. When sections from 1-8-week-old rat ovaries were labeled directly with conjugates of affinity purified anti-laminin IgG-horseradish peroxidase (HRP), the antibodies bound to all ovarian basement membranes including those surrounding follicles in different stages of maturation. In addition, intracellular labeling was seen in granulosa and theca cells of follicles undergoing rapid development (preantral and antral stages) and in basement membrane-like structures of the Call-Exner bodies. Intracellular laminin was generally not detected, however, in any cells of primordial or atretic follicles. Tissue processed for immunoelectron microscopy 1 hour after the intravenous injection of anti-laminin IgG-HRP showed binding of antibody in linear patterns along endothelial and follicular epithelial basement membranes. Discontinuous strands of laminin-positive, extracellular matrices were also seen between theca cells of all follicles. In addition, injected anti-laminin IgG labeled perisinusoidal basement membranes located within corpora luteae and patches of basement membrane material between granulosa lutein cells. When ovaries were examined 5 d after the intravenous injections of anti-laminin IgG-HRP, uneven or segmented labeling was found in subepithelial basement membranes surrounding developing follicles. Our results therefore indicate that granulosa and theca cells participate directly in basement membrane laminin biosynthesis and suggest that this new laminin is spliced into existing basement membranes during follicular growth.

Changes in Two Kinds of 20.ALPHA.-hydroxysteroid Dehydrogenase During the Luteal Phase in the Rat.

We have found previously that there are 2 kinds of 20α-hydroxysteroid dehydrogenase (20α-HSD) (EC 1.1.1.149) molecule (HSD1 and HSD2) in the mature rat ovary. In the present study, each enzyme activity was measured during pseudopregnancy (PSP) in the rat. Ovarian cytosol was collected in early (day 5), mid (day 9) or late (day 15) PSP. HSD1 activity was low in early and mid PSP and increased markedly at the end of PSP, while HSD2 activity was lower than HSD1 activity in early PSP and decreased as PSP advanced. These results suggest that the regulatory mechanism of the enzyme activity varies between HSD1 and HSD2, and that HSD1 mainly contributes to the increase in 20α-dihydroprogesterone secretion at the end of PSP. Interestingly, when ovaries at early and mid-term PSP were used, the total 20α-HSD activity in the cytosol fraction was increased markedly (163-296%) by passage through a DEAF column. This phenomenon was not observed with ovarian cytosol obtained at late PSP. Thus, we conclude that the ovary contains 2 different kinds of 20α-HSD molecule whose activities seem to be regulated separatedly, and that the HSD1 molecule is responsible for termination of the luteal phase.

Cellular localization and hormonal regulation of 7,12-dimethylbenz[ a]anthracene mono-oxygenase activity in the rat ovary

To identify the ovarian cell type(s) responsible for the metabolism of 7,12-dimethylbenz[ a]anthracene (DMBA) and their dependence on hormonal influences. DMBA mono-oxygenase activity was determined in primary cultures of cells dispersed from rat ovaries and separated by centrifugation on discontinuous Percoll TM density gradients. The contents of progesterone and oestradiol in the different cell cultures were characterized. Moreover, the morphological appearance of ovarian cells obtained from untreated and pregnant mare's serum gonadotropin (PMSG)-treated rats was examined. It is concluded from experiments with immature PMSG-treated rats, that DMBA mono-oxygenase activity is localized in follicular granulosa (and/or theca) cells. This activity decreases during luteinization, and is recovered in a population of cells harvested at a higher density on the Percoll TM gradient. The xenobiotic-metabolizing activity was not correlated to the rate of biosynthesis of progesterone or oestradiol in isolated cells, measured in the presence or absence of human chorionic gonadotropin and/or testosterone. However, a certain dependence of DBMA metabolism on steroids and/or steroid-synthesizing enzymes could not be excluded. For example, DMBA mono-oxygenase activity was markedly increased in a cell population, tentatively identified as granulosa cells, obtained from untreated mature rat ovaries upon addition of testosterone, which is the substrate for oestrogen synthesis, to the cell culture.

Purification and characterization of rat ovarian 20α-hydroxysteroid dehydrogenase

To investigate the regulatory mechanism of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) (EC 1.1.1.149) activity in ovarian tissue, the enzyme was purified from ovaries of normal mature female rats. Column chromatography of the cytosolic fraction from ovaries on DEAE-Toyopearl 650M revealed two peaks of the 20 alpha-HSD activity at different ionic strengths. These peaks were designated HSD1 and HSD2, respectively. Each of the active fractions was further purified to homogeneity by dye-affinity chromatography using Matrex Green A and AF Red-Toyopearl. Both the fractions appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (at Mr = 33,000 under reducing conditions). Under non-reducing conditions, similar values were obtained on gel-exclusion HPLC, indicating that the enzyme fractions were single-stranded, monomeric polypeptides. Homogeneous HSD1 and HSD2 were purified 361-fold and 509-fold, respectively, and differed in their substrate preference. The two enzyme fractions had Km values of 4.75 microM and 5.16 microM for 20 alpha-dihydroprogesterone, respectively, and showed almost the same RF values on reverse-phase HPLC and free-zone capillary electrophoresis. However, amino acid composition was slightly different, i.e. lysin content was higher in HSD1 than HSD2. Thus, it was clarified that two types of 20 alpha-HSD with very similar molecular structures are present in the rat ovary.

Ovarian contents of immunoreactive -endorphin and -N-acetylated opioid peptides in rats

beta-Endorphin was measured by radioimmunoassay in homogenates of ovaries from immature Sprague-Dawley rats (21-29 days of age) and found to be present at levels of about 0.6-0.7 ng/ovary. After administration of PMSG there was approximately a 4-fold increase (2-3 ng/ovary) in total ovarian immunoreactive (ir) beta-endorphin 48 h after injection. Analysis of follicular fluid from similarly treated rats indicated about the same amount of ovarian ir-beta-endorphin (2-3 ng/ovary) as in ovarian homogenates, suggesting that most of the ir-beta-endorphin is localized in follicular fluid of PMSG-primed immature rats. Immature rats were made pseudopregnant by administration of hCG 48 h after PMSG, and at 24 h after injection of hCG there was a slight, but significant and reproducible, increase in the ovarian content of ir-beta-endorphin. The serum concentration of ir-beta-endorphin was in the range of 1-3 ng/ml and was unaffected by PMSG and PMSG/hCG; likewise, the pituitary content of ir-beta-endorphin did not change following administration of gonadotrophins to immature rats. In mature cyclic animals, levels of 2-4 ng ir-beta-endorphin/ovary were found, comparable to those in the ovaries of PMSG-primed immature rats, and there were only small changes during the oestrous cycle. In addition to ir-beta-endorphin, we also obtained evidence for the presence of alpha-N-acetylated opioid peptides (endorphins or enkephalins) in the ovaries of PMSG-primed immature and mature rats. The physiological role of the opioid peptides in reproductive tissue is unknown, but they are presumably acting in an autocrine or paracrine fashion.