Cover Story:
Uterine receptivity is the specific state during early pregnancy when the endometrium is ready to accept the embryo or conceptus, and this process by which the embryo attaches to the endometrium is also known as implantation. Kubota summarized the molecular mechanisms underlying steroid hormone-induced uterine receptivity and the similarities and differences in this mechanism among different mammals (Kubota K. Molecular approaches to mammalian uterine receptivity for conceptus implantation, pp. 207–212). This review will help understand the importance of uterine receptivity and the challenges associated with alleviating implantation failure. Investigating the mechanism of uterine receptivity is necessary to develop diagnostic and therapeutic tools for successful pregnancies.
Cover Story:
Chemokines are known to regulate various reproductive functions, such as regulation of corpus luteum (CL) and uterine functions in cows. However, the role of chemokines in pregnancy has not yet been fully elucidated. Sakumoto summarized and reviewed the literature on chemokine (-receptor) expression and its physiological roles in the bovine CL and uterus during pregnancy (Sakumoto R. Role of chemokines in regulating luteal and uterine functions in pregnant cows, pp. 145–151). This review will help understand the mechanisms of chemokine-mediated interactions among the CL, uterus, immune cells, and conceptus during pregnancy in cows.
Cover Story:
The process of inducing the germ cell lineage from pluripotent stem cells, referred to as in vitro gametogenesis, aids in comprehending the mechanisms involved in germ cell differentiation and offers an alternative source of gametes for reproduction. Shirasawa et al. have developed a novel method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells (Shirasawa et al.: Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle, pp. 82–95). After a 24-hour culture with bone morphogenetic protein 4 (BMP4), followed by a three-dimensional culture with BMP4 and chemicals modulating WNT signaling, bES cells exhibited positive expression for a set of primordial germ cell (PGC) markers, including PRDM1/BLIMP1, TFAP2C, and SOX17. These outcomes are anticipated to have practical implications for the development of stem cell-based reproductive technologies in cattle.
Cover Story:
For the past century, achieving in vitro
spermatogenesis has remained a difficult challenge for researchers. In 2011,
Ogawa et al. successfully demonstrated in vitro spermatogenesis in mice using
an organ culture method. However, extending this method to other species posed
challenges for over a decade. In 2023, Ogawa’s team achieved in vitro
spermatogenesis in rats by incorporating several critical modifications to
enhance their original technique. This review presents a detailed analysis by
Ogawa et al. comparing their method with natural in vivo conditions and other
synthetic alternatives (Ogawa et al. Improvements in in vitro spermatogenesis:
oxygen concentration, antioxidants, tissue-form design, and space control, pp.
1–9). They systematically explore the merits, limitations, and inherent
constraints of the organ culture approach, delving into the specifics of medium
composition, the principles of the gas-liquid interphase method, use of
microfluidic devices, and innovation of the PDMS-ceiling method. Highlighting
the challenges faced, including regulating oxygen concentration, managing
tissue formation, and regulating culture space-control. The insights and novel
concepts shared in this review are particularly valuable for those involved in
culture or related disciplines, providing innovative content, and encouraging
further exploration in this field.
Cover Story:
Cluster of differentiation 9 (CD9) and sex-determining region Y-box 2 (SOX2) positive cells are stem/progenitor cells for hormone-producing cells in the anterior lobe (AL) of the rat pituitary gland. They are located in the marginal cell layer (MCL) facing the Rathke’s cleft between the AL and intermediate lobe (IL) and the parenchyma of the AL. Horiguchi et al. reported that CD9/SOX2-positive stem cells in the AL-side MCL have potential to supply growth hormone (GH) cells when the increase in GH cell population is required, and the AL parenchyma cells may respond to the demand of thyroid-stimulating hormone (TSH) cell supply in the adult pituitary. (Horiguchi et al. Fluctuation of CD9/SOX2-positive cell population during turnover of GH- and TSH-producing cells in adult anterior pituitary gland, pp. 308–316). These findings may provide one of the mechanisms by which hormone producing cells form from adult stem cells in the pituitary.
Cover Story:
Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction. Yamada et al. generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats (Yamada et al.; Sex difference in developmental changes in visualized Kiss1 neurons in newly generated Kiss1-Cre rats, p. 227–238). Histological analysis revealed that Kiss1 neurons, which were visualized by tdTomato, were sexually dimorphic in the anteroventral periventricular nucleus (AVPV), arcuate nucleus, and medial amygdala. As shown on the cover page, neonatal AVPV visualized Kiss1 neurons were detected only in males (upper left; compared to the AVPV in neonatal females on the upper right), but a larger number of visualized Kiss1 neurons were detected in the AVPV in females (lower right) than in males (lower left) in adulthood. The Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses on the sexual differentiation and physiological role of kisspeptin neurons.
Cover Story:
In vitro growth (IVG) culture systems provide a controlled environment for immature and developmentally incompetent oocytes to mature and acquire developmental competence in vitro. However, the reported culture periods have been longer than the follicular development in vivo. To address this, Chelenga et al. developed an 8-day IVG culture system closely resembling the in vivo follicular development period. Notably, this system yielded developmentally competent bovine oocytes when cultured in a gas-permeable culture device. In addition, the combination of the 8-day IVG culture with pre-IVM culture for IVG oocytes significantly enhanced their maturational and developmental competences (Chelenga et al. Pre-maturational culture promotes the developmental competence of bovine oocytes derived from an 8-day in vitro growth culture system, pp. 214–217). The oocyte-cumulus-granulosa complex at Days 0, 4, and 8 of IVG culture (upper left, upper right, and bottom left, respectively) and the matured IVG oocyte after pre-IVM followed by IVM cultures (bottom right) were shown on the cover page.
Cover Story:
The development of germ cells is accompanied by alterations in the cell cycle in response to external signals and intrinsic cellular mechanisms. During fetal development, male germ cells undergo G0/G1 arrest, whereas female germ cells exit the mitotic phase of the cell cycle and enter meiosis. The NANOS2 and CYP26B1 proteins in the fetal testes cause the germ cells to remain in G0/G1 arrest, which prevents them from entering the meiotic cell cycle. External signals such as RA, BMP, and WNT promote the female germ cells in the fetal ovaries to enter the meiotic phase of the cell cycle. MEIOSIN and STRA8 are transiently co-expressed in the pre-leptotene phase in spermatocytes and oocytes. The MEIOSIN-STRA8 complex ensures the establishment of the meiotic phase by activating meiotic genes in such a manner that the entry into meiosis coincides with the S phase of the cell cycle. This review discusses the development of germ cells from the viewpoint of cell cycle regulation and highlights the mechanism by which germ cells enter the meiotic phase of the cell cycle (Shimada and Ishiguro. Cell cycle regulation for meiosis in mammalian germ cells, pp. 139–146).
Cover Story:
In chickens, cryopreservation of primordial germ cells (PGCs), the embryonic precursors of gametes, is the best way to cryobank chicken germplasm. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Hamai et al. designed dimethyl sulfoxide-based and propylene glycol-based cryomedia in conjunction with trehalose and serum that achieved >50% recovery of viable PGCs after thawing while maintaining germline competency (Hamai et al. Development of cryopreservation media for the slow-freezing of cultured primordial germ cells in chicken, pp. 109–117). The offspring of Kurokashiwa, a rare chicken breed in Japan, was successfully revived from PGCs cryopreserved in this cryomedia.
Cover Story:
Nik-related protein kinase (Nrk) is an X-linked gene encoding a serine/threonine kinase belonging to GCK group 4. Nrk-knockout (Nrk-KO) mice exhibit delayed delivery. However, the mechanism of delayed labor remains largely unknown. Yomogita et al. found that serum progesterone (P4) and placental lactogen (PL-2) concentrations during late pregnancy were higher in pregnant females with Nrk-KO conceptus than in wildtype females (Yomogita et al. A possible function of Nik-related kinase in labyrinth layer of mouse placentas of delayed delivery. pp 32–40). These findings suggest that Nrk-KO mice exhibit delayed delivery due to the increase in P4 concentrations because of PL-2 hypersecretion. Moreover, Nrk was expressed in trophoblast giant cells and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is expressed in Syn-T of villi. Similar to mouse Nrk, human NRK may significantly affect placentation in evolutionary biology.
Cover Story:
NANOS3 is an evolutionarily conserved gene expressed in primordial germ cells, and it is important for germ cell development. Germ cell deletion through NANOS3 knockout has been reported in several mammalian species, although its function in pigs remains unclear. Kogasaka et al. investigated the germline effects of NANOS3 knockout in pigs (Kogasaka et al. Generation of germ cell-deficient pigs by NANOS3 knockout. p. 361–368). Transfer of gene-edited embryos using multiple gRNAs with the Cas9 protein efficiently produced NANOS3-knockout pigs. Histological analysis revealed the lack of germ cells in the testes and ovaries of all NANOS3-knockout pigs. These results demonstrated that NANOS3 is crucial for germ cell production in pigs.
Cover Story:
The on-gel culture system enables bovine blastocysts to complete cell segregation events at day (D) 10 following in vitro culture [Left panel: Epiblast marker SOX2 (Yellow), Primitive endoderm marker SOX17 (Magenta), DNA marker Hoechst (Cyan)]. This study demonstrated full-term development of D10 mature blastocysts produced using an on-gel culture system (Saito et al. Generation of viable calves derived from developmentally mature blastocysts produced by on-gel culture, pp. 330–334). This confirms that the on-gel culture system recapitulates preimplantation development to D10 and that the in vitro production of D10 blastocysts for cattle production is feasible. Two calves derived from on-gel–cultured embryos were vaginally born. Birth and placental weight were normal for both calves, and no obvious morphological abnormalities were evident. As of August 30, 2022, the calves are still alive and healthy (Right panel). The ability of the on-gel culture system to recapitulate bovine preimplantation development until D10 could contribute to studies on efficient animal production and preimplantation development in cattle.
Cover Story:
In vitro culture systems, including two-dimensional (2D) and three-dimensional (3D) culture systems, have been used to investigate various cellular functions. Present study has elucidated the effects of these culture systems on the hormonal response and cellular morphology of bovine uterine glands. The gene expression level and hormonal response of secreted proteins differed among isolated (left panels), 2D cultured (middle panels), and 3D cultured (right panels) bovine uterine glands, which might be attributed to their varied cellular morphologies (Sugino et al., Evaluation of bovine uterine gland functions in 2D and 3D culture system, pp. 254–261). Upper and lower panels show fluorescence and phase-contrast images, respectively (red: ZO-1, green: β-catenin, and blue: DAPI).
Cover Story:
Ovarian functions, such as follicular development and ovulation, are often suppressed in lactating animals. This may be a strategic adaptation to ensure the survival of lactating mothers by avoiding another pregnancy. The suppression of ovarian functions is assumed to be primarily due to the suckling-induced inhibition of hypothalamic kisspeptin neurons (the master regulators of mammalian reproductive function), followed by the inhibition of gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release. However, the mechanism mediating this inhibition is not fully understood. Sugimoto et al. demonstrated that central antagonism of somatostatin receptor 2 (SSTR2) increased kisspeptin gene expression levels in the hypothalamus of lactating rats, and that some hypothalamic glutamatergic neurons expressed SSTR2. Additionally, SSTR2 antagonism increased luteinizing hormone (LH) release in lactating rats and central glutamate receptor antagonism reversed this effect. Overall, these results suggest that central somatostatin-SSTR2 signaling, at least partly, mediates the suppression of kisspeptin gene expression and subsequent GnRH/LH release by inhibiting glutamatergic interneurons in lactating rats (Sugimoto et al. Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats. pp. 190–197).
Cover Story:
To
understand the cellular processes associated with non-invasive mode of
conceptus implantation to the maternal endometrium, Yamada et al. observed the conceptus
implantation process via adhesion of trophoblast cells to the
uterine epithelium (Yamada et al.,
Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell
formation in ovine conceptuses during the peri-implantation period, pp. 110–117).
Similar to human syncytiotrophoblast, ruminant trophoblasts form bi- and
multi-nucleated trophoblast cells. Using pregnancy associated glycoproteins
(PAGs) specific antibody, bi- and multi-nucleated trophoblast cells in cross
sections of day 21 ovine uteri (day 0 = day of estrus), including elongated
conceptuses, were observed, and it was found that bi- and multi-nucleated
trophoblast cells existed on the uterine epithelium or next to the uterine
stroma. These results suggest that the formation of bi- and multi-nucleated
trophoblast cells facilitates the placental formation in ruminants.
Cover Story:
Ogata et al. investigated the effects of reduced glutathione (GSH) supplementation in a freezing extender on frozen-thawed semen quality and subsequent in vitro fertilization (IVF) results (Ogata et al. pp. 53–61). GSH supplementation did not affect sperm viability or acrosome integrity (left panel, FITC-PNA staining) in any of the bulls, although few bulls displayed increased DNA damage (right panel, TUNEL staining). However, pronucleus formation and embryonic development after IVF improved in those bulls. These results suggest that supplementing the freezing extender with GSH may improve in vitro embryo production from frozen semen.
Cover Story:
The endometrial surface is required to reject attachment of exogenous undesirable substances such as pathogens, while occasionally shifting properties to allow physical interaction with an embryo to support a successful pregnancy. Kubota et al. reported that MUC1, a potent inhibitor of cellular contact via its large extracellular glycoprotein domain, localizes to the apical surface of bovine endometrium (upper panel, green staining). This MUC1 barrier can be removed in response to steroid signaling at receptive phase and during pregnancy (lower panel), as revealed by immunohistochemistry (p. 386–391). Red signal (pseudo-color) represents DAPI-stained nuclei. Interestingly, MUC1 expression is elevated in the endometrium of cows with a long postpartum interval, which is a known risk for reducing female fertility. Thus, this observation is consistent with the proposed anti-attachment role of MUC1. These results suggest that MUC1 regulation plays a role in endometrial homeostasis and embryo implantation, and its negative correlation with fertility may be used as a diagnostic of reproductive efficiency.
Cover Story:
Bidirectional communication between oocytes and granulosa cells or cumulus cells is maintained through transzonal projections (TZPs). These structures are important for oocyte growth, although the mechanism underlying TZP development remains unclear. Fushii et al. demonstrated the disappearance of TZPs by denudation of bovine growing oocytes and the reestablishment of TZPs by coculture of TZP-free denuded oocytes (DOs) with mural granulosa cells (MGCs) (Fushii et al. pp. 300–306). Twenty-four hours after denudation, almost all TZPs disappeared, and after coculture and subsequent growth culture of DOs with MGCs, TZPs were reestablished (upper). Additionally, the oocytes in reconstructed complexes grew fully and acquired meiotic competence, suggesting that the reestablished TZPs are able to support oocyte growth similar to those in cultured oocyte-cumulus cell-mural granulosa cell complexes (middle) and in vivo grown oocytes (lower).
During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. Morikawa et al. examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes (Morikawa et al. pp. 273–281). Oocyte-cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with GDF9 (upper left) and BMP15 (upper right) for 5 days. Only GDF9 increased the OCC diameter in a dose-dependent manner. OCCs that had been cultured with GDF9 or BMP15 were subjected to a subsequent maturation culture. Those OCCs cultured with GDF9 expanded loosely (lower left), whereas those with BMP15 expanded fully and matured to the second metaphase (lower right).
Cover Story:
In mammalian female reproduction, primordial follicles serve as stores to sustain the ovulation cycle. Regulations of primordial follicle development, activation, and dormancy in mice are summarized in a review by Nagamatsu (pp. 189–195). The importance of mechanical stress, especially extracellular matrix (ECM)-mediated pressure, for the maintenance of primordial follicle dormancy was recently demonstrated. Primordial follicles treated with collagenase, trypsin, and knockout serum replacement (KSR) (this mixture called CTK) to digest ECM were examined by immunohistochemistry. Ovaries treated solely with phosphate-buffered saline displayed primordial follicles composed of flat granulosa cells and complex stress fibers, as revealed by phalloidin. In contrast, CTK treatment resulted in fewer stress fibers and cuboidal-shaped granulosa cells, suggesting oocyte activation.