H.T.A. van Tol

Nucleotide sequences of cDNAs encoding precursors of human insulin-like growth factor II (IGF-II) and an IGF-II variant

We have isolated 3 cDNA clones encoding human IGF‐II and a variant of IGF‐II. The amino acid sequence encoded by the IGF‐II cDNA is identical to the sequence previously described [(1978) FEBS Lett. 89, 283‐286]. In the amino acid sequence predicted by the IGF‐II variant cDNA, the Ser residue 29 in the B‐domain has been replaced by an Arg‐Leu‐Pro‐Gly sequence. The corresponding mRNAs probably arise by alternative splicing of a common RNA precursor. The IGF coding region of the cDNA inserts is flanked by sequences encoding a signal peptide and a carboxy‐terminal peptide indicating that both human IGF‐II and its variant are synthesized as precursors.

Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa

Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec‐hFSH or hCG. Addition of 0.05 IU rec‐hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 μm vs. 240 μm in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group nor on cumulus expansion (246 μm vs. 240 μm in the control group). RT‐PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles.

Stimulatory effect of growth hormone on in vitro maturation of bovine oocytes is exerted through cumulus cells and not mediated by IGF-I.

A previous study reported that the addition of bovine growth hormone (bGH) during in vitro maturation of bovine oocytes accelerates nuclear maturation and stimulates subsequent embryonic development. The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) contain growth hormone receptor (GHR), and whether the stimulatory effect of GH on oocyte maturation is cumulus‐dependent and mediated by insulin‐like‐growth factor (IGF‐I). The expression of growth hormone receptor mRNA in mural granulosa cells, in cumulus cells, and in the oocyte was studied using reverse transcriptase polymerase chain reaction (RT‐PCR). To investigate the importance of cumulus cells for GH‐promoted maturation, COCs and denuded oocytes were cultured for 16 hr in M199 with or without bGH (NIH‐GH‐B18). To investigate whether GH action is mediated by IGF‐I, COCs were cultured in 1) 100 ng/ml bGH, 2) 100 ng/ml bGH plus anti‐IGF‐I, 1:100 dilution, 3) 100 ng/ml h‐IGF‐I, 4) 100 ng/ml h‐IGF‐I plus anti‐IGF‐I, 1:100 dilution, and 5) anti‐IGF‐I, 1:100 dilution. Culture was performed at 39°C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage of oocytes was assessed using 4,6‐diamino‐2‐phenyl‐indole (DAPI) staining. PCR on cDNA of mural granulosa cells, cumulus cells, and oocytes revealed that mRNA for GHR was present in all cell types. Addition of GH (100 ng/ml) to the culture medium of denuded oocytes did not affect the number of metaphase II oocytes after 16 hr, while a significant (P < 0.001) increase was observed, when COCs were cultured in the presence of GH. Addition of the antibody against IGF‐I to the culture medium completely suppressed the stimulatory effect of IGF‐I on oocyte maturation and cumulus expansion, while stimulation by GH was not affected by the antibody. It is con‐ cluded that bovine cumulus cells, mural granulosa, and oo‐ cytes express mRNA for the GH receptor. The stimulatory effect of GH on bovine oocyte maturation is dependent on the cumulus cells and is not mediated by IGF‐I. Mol. Reprod. Dev. 47:175–180, 1997.

Preimplantation bovine embryos express mRNA of growth hormone receptor and respond to growth hormone addition during in vitro development

In previous studies we demonstrated that bovine cumulus oocyte complexes (COCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in vitro maturation. The aim of this study was to investigate whether bovine zygotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether supplementation of GH during embryo culture affects embryo development. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro fertilization the embryos were cultured: (a) on a monolayer of buffalo rat liver (BRL) cells in M199 supplemented with 10% FCS and 100 ng/ml bovine GH (NIH‐GH‐B18); (b) in droplets of serum‐free BRL‐conditioned medium supplemented with 100 ng/ml GH; (c) in droplets of synthetic oviductal fluid (SOF) supplemented with 100 ng/ml GH. Cultures without GH served as controls. Embryos were scored morphologically and the efficiency of the culture system was evaluated (a) as the percentage of cleaved embryos 4 days after IVF, (b) the percentage of blastocysts on Day 9 expressed on the basis of the number of oocytes at the onset of culture, and (c) the percentage of hatched blastocysts on Day 11 expressed on the basis of the total number of blastocysts present at Day 9. For gene expression, immature (GV) and mature (MII) oocytes (as positive control), embryos with less than 8 cells, 16–32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase chain reaction (RT‐PCR) to assess the expression of mRNA of GHR. Messenger RNA for GHR was found in GV and MII oocytes and in all stages of embryo development. No mRNA for GH could be detected in early and expanded blastocysts produced in SOF medium. Immunoreactive GHR was found both in trophoblastic and embryonic cells of hatched blastocysts. Addition of 100 ng/ml GH during embryo culture on a monolayer of BRL cells in M199 supplemented with 10% FCS did not affect embryo development. However, GH (100 ng/ml) supplementation during embryo culture in droplets of serum‐free BRL conditioned medium significantly (P < 0.05) enhanced the proportion of > 8‐cell embryos. Similarly, culture of embryos in droplets of SOF medium in the presence of GH (100 ng/ml) significantly (P < 0.05) enhanced the number of > 8‐cell embryos from 53.8% in control to 70.6% in GH‐treated group. Day 9 blastocyst formation in SOF medium was also significantly (P < 0.01) increased in the presence of GH (33.9%) compared to the control (20.2%). Embryos cultured in SOF without GH rarely resulted in hatched blastocysts (0.7%). However, GH supplementation remarkably enhanced the proportion of the hatched blastocysts (13%). In conclusion, expression of GHR gene in preimplantation bovine embryos, presence of the receptor, and the beneficial effect of GH on cleavage, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development. Mol. Reprod. Dev. 57:247–255, 2000.

Messenger RNA expression and protein localization of growth hormone in bovine ovarian tissue and in cumulus oocyte complexes (COCs) during in vitro maturation

The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) obtained from 2 to 8 mm follicles synthesize growth hormone (GH) during in vitro maturation. In addition the expression of growth hormone releasing hormone receptor (GHRH‐r) in the COCs before and after in vitro maturation was investigated. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed and were prepared for immunohistochemical staining to detect the presence of GH. In addition, sections of ovary were stained to study the differential localization of GH in the ovary. At 0 and 24 hr COCs were removed and together with samples from granulosa cells and theca cells were prepared for reverse transcriptase polymerase chain reaction (RT‐PCR) to assess the expression of mRNA of GH and GHRH‐r. Within COCs, cumulus cells and oocytes showed GH immunoreactivity, while expression of GH mRNA was only found in the oocyte. At the onset of culture, oocytes and cumulus cells in the majority of COCs generally showed moderate and strong staining intensity for GH, respectively. While GH staining in the cumulus cells did hardly change during 24 hr of culture, GH staining in the oocyte was absent after 24 hr of culture in 70% of COCs. Within the ovary, GH was localized in antral follicles larger than 2 mm and no staining was found in primordial, primary and secondary follicles or in the stroma. The intensity of the staining increased with the size of the follicles. Within the follicular wall the GH was persistently observed in granulosa cells, while theca cells were occasionally negative. GH mRNA in follicular compartments was only found in the oocyte and mural granulosa cells. No GHRH‐r mRNA was found in the COCs nor in the granulosa or the stroma. In conclusion, the gradual increase of GH staining during follicular development and the consistent synthesis of GH in oocytes and granulosa cells, suggest a paracrine and/or autocrine action for GH in bovine follicular growth and oocyte maturation. The absence of mRNA for GHRH receptor in the COCs indicates that ovarian production of GH is not regulated by GHRH. Mol. Reprod. Dev. 53:398–406, 1999.

Theca cells and theca-cell conditioned medium inhibit the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa

The effect of follicular cells and their conditioned media on the FSH‐induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca‐cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell–cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis‐inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH‐mediated meiosis in oocytes of COCGs. Mol. Reprod. Dev. 51:315–321, 1998.

Immunohistochemical localization and mRNA expression of activin, inhibin, follistatin, and activin receptor in bovine cumulus‐oocyte complexes during in vitro maturation

The aim of this study was to investigate whether bovine cumulus‐oocyte complexes (COCs) synthesize activin A, inhibin, and follistatin and whether they contain activin receptor during in vitro maturation. Therefore, COCs obtained from small and medium‐sized follicles were cultured in M‐199 supplemented with 10% fetal calf serum (FCS) and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed for immunohistochemical staining to detect the expression of activin A, inhibin, follistatin, and activin receptor type II proteins. At 0 and 24 hr, COCs were removed and prepared for reverse‐transcriptase polymerase chain reaction (RT‐PCR) to assess the presence of mRNA of these proteins. It appeared that cumulus cells and oocytes express activin, follistatin, and activin receptor proteins as well as their mRNA. While expression of inhibin mRNA was found exclusively in cumulus cells, the inhibin protein was present in cumulus cells and oocytes. Immunohistochemical study both in cumulus cells and in oocytes often showed a moderate and strong staining intensity for activin and follistatin, respectively. Activin staining underwent little or no change during culture except at 24 hr of maturation, where about 60% of the oocytes showed no staining. Follistatin immunoreactivity remained strong in the majority of COCs. At the onset of culture, a spotlike inhibin staining was observed in the oocyte, which increased after 12 hr and was absent at the end of culture. Activin receptor immunoreactivity in cumulus cell membranes and oolemma increased during oocyte maturation to maximum values at the end of culture in most of the COCs. It is concluded that the consistent presence of activin and the increase in activin receptor in cumulus cells and oocytes during in vitro maturation indicate a paracrine and/or autocrine action for activin on bovine oocyte maturation. This action may be modulated by inhibin and/or follistatin. Mol. Reprod. Dev. 49:186–195, 1998.

The Kit ligand/c-Kit receptor system in goat ovaries: gene expression and protein localization

Relatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.

The effect of growth hormone on rat pre-antral follicles in vitro.

The aim of the present study was to investigate whether growth hormone (GH) has any effect on the development of cultured rat pre-antral follicles. Pre-antral follicles with a diameter between 120 microm and 160 microm were mechanically isolated from 10-day-old rat ovaries and cultured in groups for 6 days in serum-free medium without GH or with GH supplemented at concentrations of 1, 10 and 100 ng/ml, respectively. DNA content of the follicles before and after culture was measured to determine whether possible growth is due to proliferation of follicular cells. To investigate the quality of follicles cultured under different conditions, the ultrastructure of the cultured follicles was studied with transmission electron microscopy. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the expression of growth hormone receptor (GHR) in pre-antral follicles. GH, regardless of the concentration, stimulated the growth of pre-antral follicles. However, follicles cultured in medium supplemented with high-dose GH (100 ng/ml) showed a significantly lower survival rate compared with the other groups. Follicles cultured in GH-containing medium showed a better ultrastructure in comparison with those cultured in medium without GH. Remarkably, scattered cortical granules were observed in oocytes of follicles cultured in the presence of GH. With RT-PCR, the presence of the mRNA of GHR was demonstrated in pre-antral follicles. It can be concluded that GH promotes rat pre-antral follicle development in vitro and better supports the morphology of cultured pre-antral follicles. The gene expression of GHR suggests that the action of GH could be mediated by its receptors present in pre-antral follicles.

Effect of pregnancy on endometrial expression of luteolytic pathway components in the mare

Endometrial oxytocin receptors (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are central components of the luteolytic pathway in cyclic mares, and their suppression is thought to be critical to luteal maintenance during early pregnancy. We examined the effect of pregnancy on endometrial expression of potential regulators of prostaglandin (PG) F2α secretion in mares. Expression of the nuclear progesterone receptor and oestrogen receptor ERα was high during oestrus, and depressed when progesterone was elevated; the opposite applied to the membrane progesterone receptor. PTGS2 was upregulated on Day 14 of dioestrus, but not pregnancy. Although OXTR mRNA expression was not elevated on Day 14 of dioestrus, protein abundance was; this increase in OXTR protein was absent on Day 14 of pregnancy. Intriguingly, gene and protein expression for PTGS2 and OXTR increased markedly between Days 14 and 21 of pregnancy suggesting that, although initial avoidance of luteolysis during pregnancy involves their suppression, this is a transient measure that delays rather than abolishes luteolytic pathway generation. The only oxytocin-PGF2α feedback loop component downregulated on both Days 14 and 21 of pregnancy was the PGF2α receptor we propose that downregulation of the PGF2α receptor uncouples the oxytocin-PGF2α feedback loop, thereby preventing generation of the large PGF2α pulses required for luteolysis.