The influence of testosterone on the expression and function of vitamin D(3) receptor (VDR) protein in the porcine ovarian follicle.

Recently it has been shown that vitamin D(3) acting via its cognate receptor (VDR) regulates the growth, differentiation and function of female reproductive tissues including ovary. The aim of the study was to examine the effect of testosterone (T) and its antagonist 2-hydroxyflutamide (HF) on VDR protein expression and function in porcine ovarian follicles. Medium size antral follicles expressing great amount of androgen receptors and represent high steroidogenic activity were used in this research. After 6 h incubation of whole follicles with T, HF or T+HF, immunohistochemical analysis of VDR revealed its nuclear localization in granulosa and theca interna cells in control and experimental groups. The expression of VDR protein was shown as a band of 48 kDa. There were no significant differences between either experimental group and the control. T influenced the function of VDR through decreased formation of VDR/RXR (retinoid X receptor) complexes (P<0.05) in both granulosa and theca interna cells, but HF abolished this effect only in granulosa cells (P<0.05). These results suggest that androgens regulate the response of follicular cells to vitamin D3 in pigs ovary via regulation of VDR transcriptional activity.

Effect of Prenatal and Neonatal Anti-Androgen Flutamide Treatment on Aquaporin 5 Expression in the Adult Porcine Ovary.

The growth of ovarian follicles is accompanied by fluid-filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti-androgen flutamide influences androgen-dependent AQP5 expression in pre-antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post-natally. The ovaries were collected from flutamide-treated and non-treated (control) sexually mature pigs. In pre-antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre-antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.

Quantification of eggshell microstructure using X-ray micro computed tomography.

1. X-ray microcomputed tomography can be used to produce rapid, fully analysable, three-dimensional images of biological and other materials without the need for complex or tedious sample preparation and sectioning. We describe the use of this technique to visualise and analyse the microstructure of fragments of shell taken from three regions of chicken eggs (sharp pole, blunt pole and equatorial region). 2. Two- and three-dimensional images and data were obtained at a resolution of 1.5 microns. The images were analysed to provide measurements of shell thickness, the spacial density of mammillary bodies, the frequency, shape, volume and effective diameter of individual pore spaces, and the intrinsic sponginess (proportion of non-X-ray dense material formed by vesicles) of the shell matrix. Measurements of these parameters were comparable with those derived by traditional methods and reported in the literature. 3. The advantages of using this technology for the quantification of eggshell microstructural parameters and its potential application for commercial, research and other purposes are discussed.

Testosterone influences water transport in porcine granulosa cells.

The development of antral ovarian follicles entails fluid accumulation, but the mechanisms regulating water flux are unknown. Aquaporins are small, integral membrane proteins facilitating passive movement of water, some of which are known to be regulated by steroid hormones. The aim of this study was to determine whether testosterone (T) influences water transport in porcine granulosa cells. To assess water movement, the swelling of granulosa cells when moved from isotonic (319 mOsm) to hypotonic (95 mOsm) medium was measured after 12-hour pre-incubation in the presence of either testosterone (T), the antiandrogen 2-hydroxyflutamide (HF) or HF and T together. Pre-incubation with T increased the swelling of granulosa cells (p < 0.01) and this was abolished by HF (p < 0.001). Neither T nor HF affected cells in isotonic medium (319 mOsm). The results indicate that T acting via intracellular androgen receptors increases water permeability of porcine granulosa cells, probably through the regulation of aquaporin activity.

Physiological temperature variants and culture media modify meiotic progression and developmental potential of pig oocytes in vitro.

Ovarian follicles in vivo are cooler than surrounding abdominal and ovarian tissues. This study investigated whether typical follicular temperatures influence the maturation and developmental potential of pig oocytes in vitro. Oocytes were synchronised at the germinal vesicle (GV) stage and incubated at 39, 37 or 35.5 degrees C. When compared with 39 degrees C, which is often used for in vitro studies, lower temperatures delayed spontaneous progression to the metaphase I and II (MI and MII) stages of meiosis. The MII was delayed by about 12 h per degrees C. All oocytes had normal morphology. Oocytes reaching GV breakdown (GVBD) at 39 degrees C were subsequently unaffected by cooling, demonstrating thermal sensitivity during the pre-GVBD stage only. Simultaneous assay of maturation-controlling kinases (maturation promoting factor (MPF) and MAPK) showed that cooling delayed kinase activation, provided it was applied prior to GVBD. Activity profiles remained coupled to the stage of meiosis. Neither enzyme was directly thermally sensitive over this temperature range. Following in vitro fertilisation, fewer blastocysts developed from embryos derived from 35.5 or 37 degrees C oocytes as compared with those from 39 degrees C oocytes. Manipulation of fertilisation timings to allow for delayed maturation showed that over-maturing or aging at lower temperatures compromises subsequent embryo development, despite normal nuclear maturation; the GV stage was again the thermally sensitive period. Cleavage rates were improved by the culture of oocytes with follicle-stimulating hormone (FSH) at 37 but not at 35.5 degrees C. Inclusion of 20% follicular fluid in the oocyte medium restored the blastocyst rate to that seen at higher temperatures. Thus, FSH and follicular fluid may allow oocytes to achieve normal developmental potential at in vivo temperatures.

Dynamic changes in meiotic progression and improvement of developmental competence of pig oocytes in vitro by follicle-stimulating hormone and cycloheximide.

The effects of FSH, LH, and epidermal growth factor (EGF) on the dynamics of nuclear maturation and subsequent embryo development were examined in pig oocytes cultured either conventionally or after preincubation with cycloheximide (CHX). In conventional culture, FSH or EGF significantly increased the rate of attainment of metaphase II (MII) for both gilt (50.0%+/-4.2% and 54.8%+/-4.3%, respectively; control, 5.8%+/-1.8%; P<0.001) and sow (87.6%+/-3.4% and 78.8%+/-3.9%, respectively; control, 7.8%+/-2.5%; P<0.001) oocytes. Gilt oocytes treated with both FSH and EGF showed an additive response (93.7%+/-2.1%). Treatment with LH had no effect. Preincubation with CHX caused the majority (84-100%) of both gilt and sow oocytes to undergo germinal vesicle breakdown. Compared to those treated with LH and/or EGF (both>80%), fewer FSH-treated oocytes reached metaphase I (43.8%+/-5.3%, P<0.001) by 14 h and MII (48.4%+/-5.9%, P<0.001) by 24 h, although the majority (71%) did mature to MII by 36 h after removal of CHX. After in vitro fertilization, higher proportions of both CHX-pretreated and untreated, FSH-exposed oocytes cleaved (71.3%+/-2.9% and 75.3%+/-3.1%, respectively) compared with those not treated with FSH (37.7%+/-3.0% and 43.0%+/-2.9%, respectively; P<0.001). Pretreatment with CHX significantly increased blastocyst yield for both FSH-treated (32.8%+/-2.0% and 10.3%+/-1.5%, respectively; P<0.001) and untreated (16.7%+/-1.5% and 9.4%+/-1.2%, respectively; P<0.001) oocytes. Polyspermy rates were unaffected. In conclusion, pig oocytes meiotically arrested by CHX before maturation retain and improve their developmental competence. FSH stimulates nuclear maturation but slows meiotic progression.

Interaction of bovine granulosa and theca cells in a novel serum-free co-culture system.

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.

Independent activation of MAP kinase and MPF during the initiation of meiotic maturation in pig oocytes.

Mitogen-activated protein (MAP) kinase is universally activated during oocyte maturation in all vertebrates studied to date. Its role in the resumption of meiosis and in the activation of maturation-promoting factor (MPF) remains unclear, especially in domestic species such as the pig. This study aimed to clarify the temporal and causal relationships between MAP kinase and MPF during meiotic maturation, particularly during the resumption of meiosis. Pig oocytes were matured synchronously in culture by treatment with cycloheximide. Kinase activities were analysed using a sensitive in vitro double-kinase assay and the specific MAP kinase pathway inhibitor U0126. MAP kinase and MPF were activated simultaneously at the time of germinal vesicle breakdown (GVBD; 6 h after removal of cycloheximide); they reached significant activity at 7 h (P < 0.05). The activities increased in parallel during GVBD (6-10 h) and peaked when the oocytes entered metaphase I (MI; 10 h). Whereas MAP kinase remained stable at peak activity thereafter, MPF activity significantly declined during the MI-MII transition (16-20 h) but increased to a second peak at MII (22 h). MAP kinase activity in denuded and cumulus-cell enclosed oocytes was completely inhibited by 20 and 80 mmicro mol U0126 l(-1), respectively. Oocytes without detectable MAP kinase activity underwent normal GVBD in terms of nuclear morphology and timing, although later meiotic stages were abnormal. The kinetics of MPF activity during GVBD were unaffected by U0126. This study has demonstrated that MAP kinase is activated simultaneously with MPF at GVBD, but that its activation is not essential for the activation of MPF nor for the resumption of the first meiosis in pig oocytes.

Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells.

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95-100%), but distributed from GVI to GVIV (GVI 56.1 +/- 9.1%, GVII 15.3 +/- 1.4%, GVIII 21.5 +/- 7.1%, GVIV 7.1 +/- 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 +/- 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1-25 microg mL(-1) resulted in 95-100% oocytes being arrested and synchronized at GVII. With >5 microg mL(-1) CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 microg mL(-1) CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6-12 h, metaphase I (MI) at 10-18 h and anaphase I/telophase I at 16-20 h. After 12 h preincubation with 5 microg mL(-1) CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.

Follicular fluid responds endothermically to aqueous dilution.

BACKGROUND: Studies suggest that ovarian follicles are cooler than their surrounding tissues. The mechanism of this remarkable phenomenon is unclear. We postulate that endothermic reactions accompany the growth-associated hydration of follicular fluid. METHODS: We performed two types of experiment, using human and animal follicular fluids. In the first, saline (50 microl) was injected into follicular fluid (500 microl) held in an equilibrated incubator, with monitoring of sample temperature. In the second, an adiabatic microcalorimeter recorded thermal shifts after injection of buffer (10 microl) into previously dialysed samples (1.4 ml). The relevance of changes observed was assessed by mathematical modelling. RESULTS: In the incubator study, 9/17 bovine and 6/12 human fluids showed a temperature fall (0.05-0.2 degrees C). Cooling was delayed by up to 2 min but sustained for 7-25 min. Remaining fluids showed no change. In the microcalorimeter, 4/9 human, 4/6 bovine, 5/5 porcine and 1/4 equine samples showed an endothermic response. Remaining samples showed either no response (bovine) or exothermy (human, equine). Pre-concentration of human follicular fluid amplified the endothermy or reversed the exothermy. Modelling indicated that the incubator-type response was of appropriate magnitude to explain follicular hypothermy. CONCLUSION: Follicular fluid responds endothermically to aqueous dilution and may contribute to follicular cooling during growth.

Follicular fluid rheology and the duration of the ovulatory process.

The fluid dynamics of ovulation were investigated to understand the mechanical role of follicular fluid in oocyte release. A set of equations describing the flow of fluid from an evacuating follicle was derived from basic principles. These equations demonstrate that, subject to assumptions about the available pressure differential and the source of the expulsive force, the size and shape of the ovulatory orifice have the largest influences on the rate of fluid loss, although the viscosity of the fluid is also an important variable. A thorough rheological examination of pig, bovine and human follicular fluids, performed using a cone-plate viscometer, demonstrated that these fluids have complex, non-Newtonian characteristics. The fluids also undergo time-dependent and spontaneous changes in viscosity at constant shear rates; some fluids were subject to coagulation-like events. Viscosity characteristics were unrelated to broad parameters of follicle development. The models used representative viscosity values to demonstrate that variations in the rate and duration of follicle evacuation, as observed by ultrasonography, could be explained largely by variations in fluid viscosity and the characteristics of the ovulatory orifice.

Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone.

Insulin-like growth factor-1 (IGF-1) is implicated in follicle development and is considered to mediate the actions of growth hormone (GH) and gonadotrophins at the ovarian level. However, the expression and secretion of IGF-1 by the ovary are controversial, partly because of species and cell-type specificity. The present study investigated whether IGF-1 is produced by ovine granulosa cells and whether its production is regulated by GH and follicle stimulating hormone (FSH). Follicles (>/=4.0 mm) were obtained from ewes during seasonal anoestrus. Granulosa cells were cultured for a total period of 96 h in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with BSA (0.1%, w:v), transferrin (0.5 microg/ml) and testosterone (100 ng/ml). In the first set of experiments, cells were incubated in the presence of bovine calf serum (BCS) (2.5%) for the initial 48 h of culture. The cells were then cultured for the next 48 h in medium without BCS, but containing either GH (0, 2, 20, and 200 ng/ml) or FSH (0, 20, 200, and 2000 ng/ml). The medium was assayed for oestradiol (E), progesterone (P) and IGF-1. There were six wells per treatment and the experiment was carried out four times. Control granulosa cells maintained both IGF-1 and E secretion, with only low levels of progesterone output. In all experiments, both GH and FSH produced significant (P<0.001) dose-related increases in E, IGF-1 and P secretion into the medium. The maximum responses to GH (20 or 200 ng/ml) were 402% for E and 528% for IGF-1 compared with controls. The maximum responses to FSH (200 or 2000 ng/ml) were 460% for E and 514% for IGF-1. The objective of the second set of experiments was to determine the effect of the progestogenic status of cells on IGF-1 production. Granulosa cells were cultured both in the presence and absence of BCS (2.5% in the medium) during the initial 48 h of culture. For the next 48 h, cells were cultured in serum-free medium. Addition of BCS to the medium during the initial 48 h of culture stimulated progesterone production. However, it did not affect either IGF-1 or oestradiol secretion between 49 and 96 h of culture, or the cell numbers at the end of culture. In conclusion, (1) IGF-1 is secreted by granulosa cells irrespective of their progestogenic status and (2) concomitant increases in E and IGF-1 production by granulosa cells as a result of GH and/or FSH treatment suggest a role for GH and FSH in the regulation of ovarian function.

Effect of restricted food intake on production, catabolism, and effects of IGF-I and cyclic nucleotides in cultured ovarian tissue of domestic nutria (Myocastor coypus).

The aims of these in vitro experiments were to examine the effects of short-term food restriction on ovarian secretory activity and the role of IGF-I and cAMP- and cGMP-dependent intracellular mechanisms in the control of ovarian function in domestic nutria. Slices of ovary from sexually mature animals kept under conditions of normal and restricted ((1/2) of standard ration) feeding were cultured with or without IGF-I (50 ng/ml), cAMP analogues (dbcAMP and Rp-cAMPS), and cGMP analogues (8-pCPT-cGMP and Rp-8-Br-PET-cGMPS; all at 100 nM). In nonovarian cells dbcAMP activates and Rp-cAMPS inhibits protein kinase A, while 8-p-CPT-cGMP activates and RP-8-Br-PET-cGMPS inhibits protein kinase G and cGMP-gated ion channels. IGF-I release and catabolism, as well as the release of progesterone (P), estradiol (E), and cAMP by the cultures, were evaluated using RIA. IGF-I did not affect cAMP release, while each of the cAMP and cGMP analogues inhibited IGF-I release in both control and experimental groups. Fasting did not affect cAMP or IGF-I release. It partially prevented the effect of Rp-cAMPS, but not of other cyclic nucleotides, on IGF-I release and inhibited IGF-I catabolism. The Rp-cAMPS and Rp-8-Br-PET-cGMPS also inhibited IGF-I catabolism and the effects were greater with tissue from food-restricted than control animals. Ovaries from the underfed nutria secreted significantly more P and less E than those from normally fed animals. IGF-I and both cAMP analogues, given alone, did not affect P release whereas a combination of IGF-I and Rp-cAMPS increased P output in control, but not in the experimental group. The 8-pCPT-cGMP had no effect P release. Rp-8-Br-PET-cGMPS, given alone or in combination with IGF-I, dramatically increased P secretion by tissue from control but not underfed animals. Estradiol secretion by tissue from underfed animals was stimulated by IGF-I, dbcAMP, Rp-cAMPS, 8-pCPT-cGMP, and Rp-8-Br-PET-cGMPS as well as by combinations of IGF-I and Rp-cAMPS or Rp-8-Br-PET-cGMPS; these effects were not seen with control tissue. The results demonstrate that: (1) ovaries of domestic nutria secrete IGF-I, P, E, and cAMP; (2) cAMP and cGMP can influence IGF-I release and catabolism; (3) the cyclic nucleotides may have an IGF-I-mediated effect on P and E output; (4) IGF-I and cyclic nucleotides can prevent the effect of undernutrition on E, but not on P release; (5) effects of cAMP and cGMP on P and E are probably not mediated by protein kinase A, protein kinase G, or cGMP-gated ion channels; and (6) food restriction can influence ovarian IGF-I catabolism, P, and E release and modulate the effects of cyclic nucleotides and IGF-I on steroidogenesis. It is concluded that ovarian secretory activity may be regulated separately by nutrition and the cyclic nucleotide-IGF-I system, and there may be functional interrelationships between these mechanisms.

Distribution of extracellular matrix components in the developing ruminant corpus luteum: a wound repair hypothesis for luteinization.

The aim of this study was to investigate corpus luteum development by visualization of extracellular matrix proteins in the tissue at sequential stages of the luteal phase. Corpora lutea were collected from oestrus-synchronized sheep and from bovine material from an abattoir. The distributions of collagen types I and IV, fibronectin and von Willebrand factor were determined using immunohistology and semi-quantitative image analysis. During the post-ovulatory period, a fibronectin- and von Willebrand factor-rich matrix occurred centrally, adjacent to the inner parenchymal surface, whereas during early luteal development a clear border of fibronectin separated the inner parenchyma from the lumen. The inner parenchyma had abundant fibronectin initially, but the amount decreased as the rate of organ growth decreased. Over the same period, the amount of collagen type I first increased and then decreased. Collagen type I and fibronectin were less abundant in other regions of the parenchyma, and the general pattern was of slightly increasing amounts of collagen type I and decreasing amounts of fibronectin as luteal development proceeded. In contrast to earlier studies, only a small percentage of large luteal cells was found to have an associated layer of collagen type IV (presumed basal lamina). It is concluded that luteal growth and maturation require organized sequences of tissue remodelling. The central meshwork of fibronectin and von Willebrand factor and sequential deposition of collagen type I and fibronectin are strongly reminiscent of events in granulation tissue. This indicates that luteinization may be best understood as a wound repair-like process that succeeds the inflammation-like events of ovulation.

Distribution of the alpha1 to alpha6 chains of type IV collagen in bovine follicles.

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen alpha chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha1, alpha2, alpha3, alpha4, and alpha5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.

Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture.

This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro. Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium. Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion. Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity. Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1. The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture. These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1. Collagen protein, however, may be only poorly synthesized in this culture model. The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue.

Gene expression and protein distribution of collagen, fibronectin and laminin in bovine follicles and corpora lutea.

The aim of this study was to locate sites of expression and deposition of collagen, fibronectin and laminin in the bovine ovary. RNA from the granulosa and basement membrane/theca fractions of maturing follicles and from corpora lutea of the early, middle and late luteal phase was probed with cDNAs for collagen types I and IV, fibronectin and laminin. Antisera against collagens I and IV were used in western analysis of protein from follicular fluid, granulosa, basement membrane/theca and corpus luteum. Collagen subunits alpha 1(I) and alpha 2(I) were expressed in the basement membrane/theca but not in the granulosa of the follicle. They were also expressed in all luteal extracts, especially those from the early phase. Collagen alpha 2(IV) was highly expressed in the basement membrane/theca and to a lesser extent in corpora lutea. Collagen alpha 3(IV) was expressed in the granulosa, basement membrane/theca and early corpus luteum. Fibronectin 1 and laminin B2 were expressed in all tissues. Laminin B1 was expressed in all tissues except the granulosa. Collagen IV was immunodetected in all follicle extracts, with the strongest signal in the basement membrane/theca. Collagen I occurred in all luteal extracts and in the basement membrane/theca but not in follicular fluid or the granulosa. These results demonstrate tissue-specific expression of ovarian structural proteins and suggest that changes occur during the progression from follicle to corpus luteum. The production of collagen IV in the follicle wall and of collagen I in the corpus luteum is consistent with previous biochemical studies. Evidence for collagen IV in the follicular antrum suggests that the follicle wall originates from granulosa as well as theca cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbic acid and fertility.

Ascorbic acid has long been associated with fertility, but no consistent study of its mechanism of action in reproductive tissues has been made. This article considers how three of ascorbic acid's principal functions, namely its promotion of collagen synthesis, its role in hormone production, and its ability to protect cells from free radicals, may explain its reproductive actions. Data relating to both ovary and testis are reviewed since ascorbate accumulates in both tissues. Both gonads exhibit cycles of tissue remodeling and of peptide and steroid secretion that can be assumed to be ascorbate-dependent. Ascorbic acid may also prevent gametes from damage by free radicals during production and fertilization. Preliminary data on the concentrations of ascorbic acid in serum and follicular fluid from women undergoing in vitro fertilization are presented. They suggest that the supply of ascorbic acid to the ovary might be a limiting factor in the ability of the preovulatory follicle to grow in response to gonadotropin stimulation. It is concluded that ascorbic acid is a key compound in gonadal physiology on which further research is needed and that a reappraisal of its potential clinical value in the treatment of various types of male and female infertility would be timely.

Potential leukocyte attractants in the bovine peri-ovulatory ovary.

This study investigated interrelationships between the bovine ovarian cycle and white blood cells and tested the hypothesis that the ovary produces collagen-like materials with leukocyte attractant activity. We examined the in vitro secretion of leukocyte attractant activity by peri-ovulatory ovarian tissues and evaluated the leukocyte attractant potential of some ovarian biochemicals. Fluid from mature ovarian follicles and medium conditioned by follicular tissue, early luteal tissue or granulosa cells had significant attractant activity. The activity could be removed by protein precipitation but not by collagenase. Collagenase also failed to alter the electrophoretic profile of the samples. Collagenase (800 IU/ml), ascorbic acid (10-1,000 micrograms/ml) and CaCl2 (50-560 micrograms/ml) had significant leukocyte attractant effects. Native collagen types I and IV (100-1,000 micrograms/ml) had fewer expressed attractant activities, which were unaffected by collagenase pre-treatment. The attractant activity of collagenase itself was removed by protein precipitation. Our observations suggest: (1) that follicular and luteal tissues produce leukocyte attractant(s); (2) that granulosa cells contribute to the secretion of this material; (3) that the principal ovarian attractants are neither the native collagen types I or IV nor their collagenase-releasable fragments; and (4) that collagenase, ascorbic acid and Ca2+ are strong candidates as attractant constituents of ovarian secretions.