Primate follicular development and oocyte maturation in vitro.

The baboon is another nonhuman primate model used for studies related to women's reproductive health in the areas of contraception, reproductive aging, infertility, implantation, and endometriosis ( D'Hooghe, 2004 ). Only a few reports have applied the baboon model to understand early events of folliculogenesis ( Fortune, 1998 ; Wandji, 1997 ). In a recent study, baboon preantral follicles were cultured in a semidegradable 3D matrix to investigate the effect of gonadotropin on follicle survival, growth, and oocyte maturation ( Xu, 2011b ). Primate ovarian tissues have denser connective tissue than rodents, which renders the isolation of individual follicles somewhat difficult without enzymatic treatments. However, collagenase digestion not only loosens the connective tissue surrounding the follicle, but may also disrupt the basement membrane and remove most, if not all, theca-interstitial cells of follicles. Whether and how the digestion itself or loss of the basement membrane and theca-interstitial cells, or combined forces would impact follicle growth in vitro is open for debate ( Abir, 1997 ; Roy, 1993 ). Initial studies on macaque ( Xu, 2009b ) and baboon ( Xu, 2011b ) follicles indicate that preantal follicles can survive and grow after collagenase treatment. However, due to variation among animals, it was difficult to uniformly control the level of stromal digestion that many times led to secondary follicle damage during isolation (unpublished results). Whether and how theca-interstitial cells promote primate follicle growth and oocyte maturation during culture awaits further study. Soft hydrogels provide a more permissive environment for follicle growth relative to rigid hydrogels ( Xu, 2006b ). Alginate hydrogels, which are not degradable and thus have a relatively stable elastic modulus, may resist the large deformations associated with significant increases in follicle diameter, which could result in a non-permissive condition for primate follicles, as they need to grow to much larger sizes than mouse follicles. A semidegradable matrix containing fibrin, alginate, and Matrigel (FAM) was employed to grow baboon preantral follicles because a previous study indicated that it provided a dynamic mechanical environment that promoted mouse follicle growth and increased the number of meiotically competent oocytes relative to alginate ( Shikanov, 2009 ). Indeed, the FAM matrix facilitated baboon follicle expansion while maintaining antral follicle architecture. Moreover, compact COCs isolated from baboon antral follicles underwent IVM to yield oocytes that reinitiated meiosis (MII stage) with a normal appearing spindle structure ( Figure 9.4 ). A necessity for FSH in mouse ( Abir, 1997 ), macaque ( Xu, 2011a ), and human ( Adriaens, 2004 ) preantral follicle development in vitro has been established. Interestingly, in the baboon, the transition from preantral to small antral follicles in vitro appears to be FSH-independent under certain culture conditions. The absence of exogenous FSH did not affect follicle survival and health in the baboon ( Xu, 2011b ), while exogenous FSH did impact follicle growth rate, particularly in the beginning of culture. With a higher dose of FSH (100 mIU/ml), follicles increased from an average diameter of 288 ± 9 μm to 439 ± 24 μm in 4 days, while it took 8-10 days to reach an equivalent size when follicles were grown in the absence of or with a lower dose of FSH (10 mIU/ml). Although follicles exposed to a higher dose of FSH showed a faster growth rate in the beginning of culture, growth plateaued after antral formation. On the other hand, the follicles cultured without FSH steadily grew to an equivalent diameter and formed an antral cavity. Whether the FSH-independent growth of baboon preantral follicles, unlike that of macaque or human follicles, is due to species differences or different culture techniques (e.g., presence of Matrigel, and its associated growth factors, as an extracellular matrix) is unknown.

Ovarian follicular development is a dynamic process that is regulated by complex interactions between gonadotropic hormones and local paracrine/autocrine factors ( Gougeon, 1996 ). Although progress in understanding early folliclulogenesis has been made, particularly in mice through gene manipulation ( Matzuk, 2000 ; Drummond, 2006 ), the regulation and dynamics of primate folliculogenesis, aside from phenotypic analysis in women ( Chand, 2010 ; Ewens, 2010 ), remain poorly understood due to the lack of adequate in vitro models. Two general approaches of follicle culture have been pursued with dissected follicles attaching to the culture plate and growing two-dimensionally (2D), or follicles encapsulated in a matrix that maintains their intact three-dimensional (3D) structure. Secondary follicles from marmosets produced metaphase II (MII) oocytes following 2D culture and oocyte in vitro maturation (IVM; Nayudu, 2003 ). Human preantral follicles encapsulated in agar ( Roy, 1993 ) or collagen ( Abir, 1997 , 1999 , 2001 ) gels maintained their morphology and grew to the early antral stage. Recently, early antral follicles were obtained from ovarian cortical strip culture ( Telfer, 2008 ). Biomaterials have been applied to 3D follicle culture, which maintain the cell-cell and cell-matrix connections important in regulating follicle development in vivo ( West, 2007 ). Alginate was successfully used for the culture of murine follicles, and its application to nonhuman primates resulted in the growth of small preantral follicles through the antral stage with production of ovarian steroids and local factors, as well as oocyte maturation ( Xu, 2009a , 2010 , 2011a , 2011b ). This in vitro follicle maturation (IFM) technique is a powerful instrument for monitoring the endocrine and paracrine/autocrine function of individual follicles, as well as manipulating regulatory factors or signaling pathways, which is essential to obtain knowledge of their role(s) and importance in follicular and oocyte development in primates. Besides the research relevance to basic ovarian biology, IFM, combined with advances in ovarian tissue cryopreservation ( Ting, 2011 , 2012 ), may be applied to fertility preservation in women, including cancer patients ( Jeruss, 2009 ). Although ovarian cortex transplantation using fresh tissue in monkeys ( Lee, 2004 ), as well as using fresh and cryopreserved tissue in women ( Dittrich, 2012 ; Donnez, 2004 ; Silber, 2008 ), yielded viable offspring, the IFM approach has the advantage of eliminating the reintroduction of cancer cells into the patients and providing a way to harvest more mature oocytes ( Woodruff, 2007 ). Live offspring were generated in mice through IFM ( Xu, 2006a ), and studies demonstrated the potential application of IFM in humans ( Xu, 2009b ; Smitz, 2010 ). Even though the meiotic competence and developmental capacity of human oocytes grown from preantral stages in vitro have not yet been reported, animal studies indicate that ovarian tissue storage followed by IFM is a valid prospect for clinical translation to humans to prevent the destruction or damage to ovarian germline cells caused by radiotherapy and/or chemotherapy ( Woodruff, 2007 ). Information obtained by growing rhesus macaque and baboon follicles during 3D culture may be valuable in identifying the optimal conditions for primate follicle culture prior to human application. Thus, the current status of efforts to study primate follicular development during IFM are summarized to (1) consider the characteristics and regulation of the survival and growth of primate preantral follicles during encapsulated 3D culture; (2) review the endocrine (ovarian steroid hormones), paracrine/autocrine (local factors), and gametogenic (oocyte maturation) function of primate follicles prior to and during antral development in vitro ; and (3) discuss the challenges and opportunities for further advances using primate follicles and cumulus-oocyte complexes (COCs) for fertility preservation.

Advances in the 3D culture allow primate secondary follicles to grow to the small antral stage and yield mature oocytes. The following conditions to optimize culture are now being employed: 1) a higher dose (3 ng/ml) of FSH for the first 3 weeks to support follicle survival, followed by a low dose (0.3 ng/ml) to avoid premature differentiation (luteinization); 2) a low concentration (0.5 mg/ml) of fetuin to maintain alginate gel integrity; and 3) low O2 tension at 5% to limit detrimental effects of high oxygen on follicle survival and mimic the follicular environment in vivo . In the presence of a higher dose of FSH, growing follicles reach the multilayer stage and then form an antrum. After switching to low-dose FSH, fast-grow follicles continue to grow until the diameters are over 1 mm, when some can respond to hCG to yield MII oocytes. AMH production increases when follicles are at the multilayer stage. Steroid and VEGF levels are elevated around or after antrum formation while AMH level decreases ( Figure 9.5 ). But under the best case scenario, some follicles do not survive, and those that survive vary in growth potential. Healthy oocytes are obtained from in vitro -developed antral follicles, but few mature to the stage of spontaneous reinitiation of meiosis after removal from the follicle, or in response to hCG. The IFM protocol needs further improvement to produce more meiotically and developmentally competent oocytes for subsequent embryonic development after fertilization. Studies can be conducted using nonhuman primates to compare the structure and function between SAFs or their COCs derived from culture in various matrices and those developed in vivo during spontaneous menstrual cycles. These studies will be valuable for assessing whether the encapsulated 3D system allows coordinated development of granulosa and theca cells, plus cumulus cells and oocytes, similar to that in vivo , and if not, will help define cellular functions that require further optimization in the culture system. This culture system also provides a way to examine the function of endocrine/paracrine factors during folliculogenesis in primates, including gene and protein expression, as well as metabolic pathways. This information can be used to discover biomarkers that predict or monitor follicle and/or oocyte condition during IFM. Since tissue resources from nonhuman primates or women are limited, efforts are warranted to more efficiently use of the entire follicle pool. Smaller resting primordial and early growing primary follicles represent a larger follicle population than secondary follicles. Primordial follicles within pieces of the baboon ( Wandji, 1997 ) or human ( Telfer, 2008 ) ovarian cortex can survive and develop to the secondary stage in serum-free culture. Human ( Vanacker, 2011 ) and macaque ( Hornick, 2012 ) primordial or primary follicles can be isolated and maintain their viability when cultured in groups. To date, efforts to grow and mature individual primordial and primary follicles in vitro have not been reported in primates, especially under chemically-defined conditions. Preliminary experiments conducted in rhesus macaques indicate that it is possible to grow individual primary follicles (80-120 μm in diameter) in vitro to the small antral stage, which function in steroidogenesis, local factor production, and oocyte maturation ( Xu, 2011c ). However, the culture interval required to reach the small antral stage is longer when starting with primary verse secondary follicles (13 vs. 5 weeks). There are also SAFs that range in size from 0.5-1.5 mm in diameter in the medullary region of the ovary. COCs obtained from these follicles are able to achieve cumulus expansion and oocyte meiotic maturation after IVM in both rhesus macaques ( Peluffo, 2010 ) and baboons ( Xu, 2011b ), with demonstration of fertilization and early embryonic development in vitro to the expanded blastocyst stage ( Peluffo, 2012 ). IFM in nonhuman primates is a powerful tool to improve the understanding of the basic biology of primate follicles, such as the heterogeneity of the preantral follicle pool, role(s) of ovarian steroids and local factors on folliculogenesis and oocyte developmental capacity. Once achieved, this knowledge may be valuable in identifying optimal conditions for human follicle culture, with the ultimate goal of translating the experimental results and products to patients, thereby facilitating diagnostic and therapeutic approaches for female fertility.

Pilot studies were conducted, using the rhesus macaque as a model, to compare gene expression profiles between SAFs (∼1 mm in diameter) derived from encapsulated 3D culture and those developed in vivo during the early follicular phase of spontaneous menstrual cycles (unpublished data). Preliminary data, generated from Affymetrix microarray assays, indicated that the mRNA levels from genes of major steroidogenic enzymes did not differ (e.g., steroid 17-alpha-hydroxylase/17,20 lyase and aromatase), except that low density lipoprotein receptor ( LDLR ) was up-regulated in cultured SAFs compared to those developed in vivo ( Table 9.1 ). The increase in LDLR mRNA expression may be due to the prolonged exposure of cultured follicles to exogenous FSH, which is consistent with the observation that FSH increased both LDLR mRNA ( LaVoie, 1999 ) and protein ( Veldhuis, 1988 ) expression in cultured porcine granulosa cells. The mRNAs for some local factors secreted by SAFs, e.g., AMH and AMH receptor, were not expressed differently between in vitro - and in vivo -developed SAFs. However, mRNAs for the angiogenic factor VEGF and its receptors were down regulated in cultured SAFs ( Table 9.1 ), which indicates that, though with similar sizes, the cultured SAFs may not achieve the same maturation state as in vivo -derived SAFs that requires vascularization for further development ( Stouffer, 2001 ). When analyzing factors involved in cell death, the mRNAs from genes encoding caspases or autophagy-related proteins (e.g., Autophagy related 7 and Beclin 1) did not differ between in vitro - and in vitro -developed SAFs. In contrast, mRNA expression for anti-apoptosis factors increased in cultured SAFs compared to in vivo -derived SAFs, including glutamate-cysteine ligase catalytic subunit ( GCLC ) and epidermal growth factor receptor ( EGFR ) ( Table 9.1 ). Exogenous FSH in the culture media may promote the GCLC and EGFR mRNA expression, as reported in rat SAF and granulosa cell culture ( Hoang, 2009 ; Fujinaga, 1994 ). Thus, macaque SAFs derived from encapsulated 3D culture exhibited some similarities as well as differences in gene expression compared to those of in vivo -developed SAFs. Further experiments are warranted to validate the microarray results, and to consider the causes and effects of altered gene expression as clues to improve coordinated follicular development leading to oocyte competence.