F. Izadyar

Proliferation and Differentiation of Bovine Type A Spermatogonia During Long-Term Culture

Abstract The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2–4 wk at 32°C or 37°C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37°C than at 32°C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation.

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH‐GH‐B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39°C in a humidified atmosphere with 5% CO2 in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6‐diamino‐2‐phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm‐binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm‐binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation. Mol. Reprod. Dev. 49:444–453, 1998.

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.

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.

Spermatogonial stem cell transplantation

The development of the spermatogonial transplantation technique has given new impetus to research on spermatogonial stem cells. Possibilities opened by this technique include: (a) New ways to study fundamental aspects of spermatogenesis; (b) Generation of transgenic large domestic animals; (c) Protection of (young) male cancer patients from infertility due to chemotherapy or radiotherapy. Spermatogonial stem cell transplantation for the above purposes encompasses a number of steps. First, the stem cells have to be isolated and possibly purified. Second, it should be possible to cryopreserve the stem cells, for example till the children have reached puberty. Third. it should be possible to culture spermatogonial stem cells for a prolonged period of time which would also allow transfection and subsequent selection of stably transfected cells. Fourth, in case of animal studies. the host testis should be emptied from endogenous stem cells. This is probably best done by local irradiation. Finally, the stem cells will have to be transplanted.

Effect of Growth Hormone Releasing Hormone (ghrh) and Vasoactive Intestinal Peptide (VIP) on in vitro bovine oocyte maturation

The aim of this study was to investigate the effects of growth hormone releasing hormone (GHRH) and the structural-related peptide vasoactive intestinal peptide (VIP) on nuclear maturation, cortical granule distribution and cumulus expansion of bovine oocytes. Bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of either 100 ng/mL bovine GHRH or 100 ng/mL porcine VIP. The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage was assessed after 16 or 24 h of incubation using DAPI staining. Cortical granule distribution was assessed after 24 h of incubation using FITC-PNA staining. To assess the effects of GHRH and VIP on cumulus expansion, COCs were incubated for 24 h under the conditions described above. In addition, 0.05 IU/mL recombinant human FSH was added to GHRH and VIP groups. Cultures without GHRH/VIP/FSH or with only FSH served as negative and positive controls, respectively. At 16 h neither GHRH (42.9%) nor VIP (38.5%) influenced the percentage of MII stage oocytes compared with their respective controls (44.2 and 40.8%). At 24 h there also was no difference in the percentage of MII oocytes between GHRH (77.0%), VIP (75.3%) and their respective controls (76.0 and 72%). There was no significant cumulus expansion in the GHRH or VIP group, while FSH induced significant cumulus expansion compared with the control groups, which were not inhibited by GHRH or VIP. Distribution of cortical granules was negatively affected by GHRH and VIP. The percentage of oocytes showing more or less evenly dispersed cortical granules in the cortical cytoplasm aligning the oolemma (Type 3) was lower in the GHRH (2.7%) and VIP (7.8%) groups than in the control group (15.9%). In conclusion, GHRH and VIP have no effect on nuclear maturation or cumulus expansion of bovine COCs but retard cytoplasmic maturation, as reflected by delayed cortical granule migration.

Spermatogonial Stem Cell Development

Spermatogonial stem cells originate from primordial germ cells (PGCs) that derive from epiblast cells (embryonal ectoderm) (Lawson and Pederson 1992). During fetal development the PGCs proliferate and migrate to the genital ridges, where they become enclosed in the seminiferous cords formed by Sertoli cell precursors. Once in the seminiferous cords, the cells are called gonocytes which are morphologically different from the PGCs (Clermont and Perey 1957; Huckins and Clermont 1968; Sapsford 1962). Gonocytes proliferate for a while and then become quiescent. In mice and rats, gonocytes start spermatogenesis shortly after birth and give rise to spermatogonial stem cells as well as the first Al spermatogonia (review de Rooij 1998).